Modified Short Interfering Nucleic Acid (siNA) Molecules and Uses Thereof

ABSTRACT

Disclosed herein are short interfering nucleic acid (siNA) molecules comprising modified nucleotides and uses thereof. The siNA molecules may be double stranded and comprise modified nucleotides selected from 2′-O-methyl nucleotides and 2′-fluoro nucleotides. Further disclosed herein are siNA molecules comprising (a) a phosphorylation blocker, conjugated moiety, or 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.17/672,268, filed Feb. 15, 2022, which is a Continuation of U.S.application Ser. No. 17/194,079, filed Mar. 5, 2021, which claimspriority to U.S. Provisional Application No. 62/986,150, filed Mar. 6,2020, and U.S. Provisional Application No. 63/109,196, filed Nov. 3,2020, the disclosures of which are hereby incorporated by reference intheir entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML. format and is hereby incorporated byreference in its entirety. Said XML. copy, created on 15 Nov., 2022, isnamed 122400-0307_SL.XML. and is 768000 bytes in size.

FIELD OF THE INVENTION

Described are short interfering nucleic acid (siNA) molecules comprisingmodified nucleotides, compositions, and uses thereof.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a biological response to double-stranded RNAthat mediates resistance to both endogenous parasitic and exogenouspathogenic nucleic acids, and regulates the expression of protein-codinggenes. The short interfering nucleic acids (siNA), such as siRNA, havebeen developed for RNAi therapy to treat a variety of diseases. Forinstance, RNAi therapy has been proposed for the treatment of metabolicdiseases, neurodegenerative diseases, cancer, and pathogenic infections(See e.g., Rondindone, Biotechniques, 2018, 40(4S),doi.org/10.2144/000112163, Boudreau and Davidson, Curr Top Dev Biol,2006, 75:73-92, Chalbatani et al., Int J Nanomedicine, 2019,14:3111-3128, Arbuthnot, Drug News Perspect, 2010, 23(6):341-50, andChernikov et. al., Front. Pharmacol., 2019,doi.org/10.3389/fphar.2019.00444, each of which are incorporated byreference in their entirety). However, major limitations of RNAi therapyare the ability to effectively deliver siRNA to target cells and thedegradation of the siRNA.

The present disclosure improves the delivery and stability of siNAmolecules by providing siNA molecules comprising modified nucleotides.The siNA molecules of the present disclosure provide optimizedcombinations and numbers of modified nucleotides, nucleotide lengths,design (e.g., blunt ends or overhangs, internucleoside linkages,conjugates), and modification patterns for improving the delivery andstability of siNA molecules.

SUMMARY OF THE INVENTION

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising: (a) a sense strand comprising a first nucleotide sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%identical to an RNA corresponding to a target gene, wherein the firstnucleotide sequence: (i) is 15 to 30 nucleotides in length; and (ii)comprises 15 or more modified nucleotides independently selected from a2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least onemodified nucleotide is a 2′-O-methyl nucleotide and the nucleotide atposition 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end ofthe first nucleotide sequence is a 2′-fluoro nucleotide; and (b) anantisense strand comprising a second nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to the RNA corresponding to the target gene, wherein thesecond nucleotide sequence: (i) is 15 to 30 nucleotides in length; and(ii) comprises 15 or more modified nucleotides independently selectedfrom a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein atleast one modified nucleotide is a 2′-O-methyl nucleotide and at leastone modified nucleotide is a 2′-fluoro nucleotide.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising: (a) a sense strand comprising a first nucleotide sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%identical to an RNA corresponding to a target gene, wherein the firstnucleotide sequence: (i) is 15 to 30 nucleotides in length; and (ii)comprises 15 or more modified nucleotides independently selected from a2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least onemodified nucleotide is a 2′-O-methyl nucleotide and at least onemodified nucleotide is a 2′-fluoro nucleotide; and (b) an antisensestrand comprising a second nucleotide sequence that is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNAcorresponding to the target gene, wherein the second nucleotidesequence: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15or more modified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide, wherein at least one modifiednucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 2,5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of the secondnucleotide sequence is a 2′-fluoro nucleotide.

In some embodiments, the first nucleotide sequence comprises 16, 17, 18,19, 20, 21, 22, 23, or more modified nucleotides independently selectedfrom a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. In someembodiments, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleotides inthe first nucleotide sequence are modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. Insome embodiments, between 2 to 15 modified nucleotides of the firstnucleotide sequence are 2′-fluoro nucleotides. In some embodiments,between 2 to 10 modified nucleotides of the first nucleotide sequenceare 2′-fluoro nucleotides. In some embodiments, between 2 to 6 modifiednucleotides of the first nucleotide sequence are 2′-fluoro nucleotides.In some embodiments, at least 2, 3, 4, 5, or 6 modified nucleotides ofthe first nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2modified nucleotides of the first nucleotide sequence are 2′-fluoronucleotides. In some embodiments, between about 2 to 25 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 2 to 20 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 5 to 25 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 10 to 25 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 12 to 25 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, or 22 modified nucleotides of the first nucleotidesequence are 2′-O-methyl nucleotides. In some embodiments, less than orequal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotidesequence are 2′-O-methyl nucleotides.

In some embodiments, the second nucleotide sequence comprises 16, 17,18, 19, 20, 21, 22, 23, or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. Insome embodiments, 70%, 75%, 80%, 85%, 90%, 95% or 100% of thenucleotides in the second nucleotide sequence are modified nucleotidesindependently selected from a 2′-O-methyl nucleotide and a 2′-fluoronucleotide. In some embodiments, between 2 to 15 modified nucleotides ofthe second nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, between 2 to 10 modified nucleotides of the secondnucleotide sequence are 2′-fluoro nucleotides. In some embodiments,between 2 to 6 modified nucleotides of the second nucleotide sequenceare 2′-fluoro nucleotides. In some embodiments, at least 2, 3, 4, 5, or6 modified nucleotides of the second nucleotide sequence are 2′-fluoronucleotides. In some embodiments, less than or equal to 10, 9, 8, 7, 6,5, 4, 3, or 2 modified nucleotides of the second nucleotide sequence are2′-fluoro nucleotides. In some embodiments, between about 2 to 25modified nucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 2 to 20 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 5 to 25 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 10 to 25 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 12 to 25 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, or 22 modified nucleotides of the second nucleotidesequence are 2′-O-methyl nucleotides. In some embodiments, less than orequal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,or 2 modified nucleotides of the second nucleotidesequence are 2′-O-methyl nucleotides.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising: (a) a sense strand comprising a first nucleotide sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%identical to an RNA corresponding to a target gene, wherein the firstnucleotide sequence: (i) is 15 to 30 nucleotides in length; (ii)comprises 15 or more modified nucleotides independently selected from a2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least onemodified nucleotide is a 2′-O-methyl nucleotide and at least onemodified nucleotide is a 2′-fluoro nucleotide; and (iii) comprises 1 ormore phosphorothioate internucleoside linkage; and (b) an antisensestrand comprising a second nucleotide sequence that is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNAcorresponding to the target gene, wherein the second nucleotidesequence: (i) is 15 to 30 nucleotides in length; (ii) comprises 15 ormore modified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide, wherein at least one modifiednucleotide is a 2′-O-methyl nucleotide and at least one modifiednucleotide is a 2′-fluoro nucleotide; and (iii) comprises 1 or morephosphorothioate internucleoside linkage.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising: (a) a sense strand comprising a first nucleotide sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%identical to an RNA corresponding to a target gene, wherein the firstnucleotide sequence: (i) is 15 to 30 nucleotides in length; and (ii)comprises 15 or more modified nucleotides independently selected from a2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least onemodified nucleotide is a 2′-O-methyl nucleotide and at least onemodified nucleotide is a 2′-fluoro nucleotide; and (b) an antisensestrand comprising a second nucleotide sequence that is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNAcorresponding to the target gene, wherein the second nucleotidesequence: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15or more modified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide, wherein at least one modifiednucleotide is a 2′-O-methyl nucleotide and at least one modifiednucleotide is a 2′-fluoro nucleotide, wherein the siNA further comprisesa phosphorylation blocker, a galactosamine, or 5′-stabilized end cap.

In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 nucleotides atposition 3, 5, 7, 8, 9, 10, 11, 12, and/or 17 from the 5′ end of thefirst nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at position 3 from the 5′ end of the firstnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, thenucleotide at position 5 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 7 from the 5′ end of the first nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, the nucleotide at position 8from the 5′ end of the first nucleotide sequence is a 2′-fluoronucleotide. In some embodiments, the nucleotide at position 9 from the5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. Insome embodiments, the nucleotide at position 12 from the 5′ end of thefirst nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at position 17 from the 5′ end of the firstnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments,nucleotide at position 10 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 11 from the 5′ end of the first nucleotide sequence is a2′-fluoro nucleotide.

In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotidesat position 2, 5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of thesecond nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at position 2 from the 5′ end of the secondnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, thenucleotide at position 5 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 6 from the 5′ end of the second nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, the nucleotide at position 8from the 5′ end of the second nucleotide sequence is a 2′-fluoronucleotide. In some embodiments, the nucleotide at position 10 from the5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide. Insome embodiments, the nucleotide at position 14 from the 5′ end of thesecond nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotides at position 16 from the 5′ end of thesecond nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at position 17 from the 5′ end of the secondnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, thenucleotide at position 18 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

In some embodiments, the nucleotides in the second nucleotide sequenceare arranged in an alternating 1:3 modification pattern, and wherein 1nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methylnucleotides. In some embodiments, the alternating 1:3 modificationpattern occurs 2-5 times. In some embodiments, at least two of thealternating 1:3 modification pattern occur consecutively. In someembodiments, at least two of the alternating 1:3 modification patternoccurs nonconsecutively. In some embodiments, at least 1, 2, 3, 4, or 5alternating 1:3 modification pattern begins at nucleotide position 2, 6,10, 14, and/or 18 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 2 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 6 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 10 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 14 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 18 from the 5′ end of the antisense strand.

In some embodiments, the nucleotides in the second nucleotide sequenceare arranged in an alternating 1:2 modification pattern, and wherein 1nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methylnucleotides. In some embodiments, the alternating 1:2 modificationpattern occurs 2-5 times. In some embodiments, at least two of thealternating 1:2 modification pattern occurs consecutively. In someembodiments, at least two of the alternating 1:2 modification patternoccurs nonconsecutively. In some embodiments, at least 1, 2, 3, 4, or 5alternating 1:2 modification pattern begins at nucleotide position 2, 5,8, 14, and/or 17 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 2 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 5 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 8 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 14 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 17 from the 5′ end of the antisense strand.

Disclosed herein is a short interfering nucleic acid (siNA) moleculerepresented by Formula (VIII):

5′-A_(n) ¹B_(n) ²A_(n) ³B_(n) ⁴A_(n) ⁵B_(n) ⁶A_(n) ⁷B_(n) ⁸A_(n) ⁹-3′3′-C_(q) ¹A_(q) ²B_(q) ³A_(q) ⁴B_(q) ⁵A_(q) ⁶B_(q) ⁷A_(q) ⁸B_(q) ⁹A_(q)¹⁰B_(q) ^(1l)A_(q) ¹²-5′wherein:

-   -   the top strand is a sense strand comprising a first nucleotide        sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,        90%, 95%, or 100% identical to an RNA corresponding to a target        gene, wherein the first nucleotide sequence comprises 15 to 30        nucleotides; the bottom strand is an antisense strand comprising        a second nucleotide sequence that is at least about 60%, 65%,        70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA        corresponding to the target gene, wherein the second nucleotide        sequence comprises 15 to 30 nucleotides;    -   each A is independently a 2′-O-methyl nucleotide or a nucleotide        comprising a 5′-stabilized    -   end cap or a phosphorylation blocker;    -   B is a 2′-fluoro nucleotide;    -   C represents overhanging nucleotides and is a 2′-O-methyl        nucleotide;    -   n¹=1-4 nucleotides in length;    -   each n², n⁶, n⁸, q³, q⁵, q⁷, q⁹, q¹¹, and q¹² is independently        0-1 nucleotides in length; each n³ and n⁴ is independently 1-3        nucleotides in length;    -   n⁵ is 1-10 nucleotides in length;    -   n⁷ is 0-4 nucleotides in length;    -   each n⁹, q¹, and q² is independently 0-2 nucleotides in length;    -   q⁴ is 0-3 nucleotides in length;    -   q⁶ is 0-5 nucleotides in length;    -   q⁸ is 2-7 nucleotides in length; and    -   q¹⁰ is 2-11 nucleotides in length.

Disclosed herein is a short interfering nucleic acid (siNA) moleculerepresented by Formula (IX):

5′-A₂₋₄B₁A₁₋₃B₂₋₃A₂₋₁₀B₀₋₁A₀₋₄B₀₋₁A₀₋₂-3′3′-C₂A₀₋₂B₀₋₁A₀₋₃B₀₋₁A₀₋₅B₀₋₁A₂₋₇B₁A₂₋₁₁B₁A₁-5′wherein:

-   -   the top strand is a sense strand comprising a first nucleotide        sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,        90%, 95%, or 100% identical to an RNA corresponding to a target        gene, wherein the first nucleotide sequence comprises 15 to 30        nucleotides; the bottom strand is an antisense strand comprising        a second nucleotide sequence that is at least about 60%, 65%,        70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA        corresponding to the target gene, wherein the second nucleotide        sequence comprises 15 to 30 nucleotides;    -   each A is independently a 2′-O-methyl nucleotide or a nucleotide        comprising a 5′-stabilized end cap or a phosphorylation blocker;    -   B is a 2′-fluoro nucleotide;    -   C represents overhanging nucleotides and is a 2′-O-methyl        nucleotide.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising (a) a sense strand comprising a first nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are atpositions 3, 7-9, 12, and 17 from the 5′ end of the first nucleotidesequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2,4-6, 10, 11, and 13-16 from the 5′ end of the first nucleotide sequence;and (b) an antisense strand comprising a second nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are atpositions 2 and 14 from the 5′ end of the second nucleotide sequence,and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17from the 5′ end of the second nucleotide sequence. In some embodiments,the first nucleotide sequence consists of 19 nucleotides. In someembodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the5′ end of the first nucleotide sequence. In some embodiments, the secondnucleotide sequence consists of 21 nucleotides. In some embodiments,2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of thesecond nucleotide sequence.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising (a) a sense strand comprising a first nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are atpositions 3, 7, 8, and 17 from the 5′ end of the first nucleotidesequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2,4-6, and 9-16 from the 5′ end of the first nucleotide sequence; and (b)an antisense strand comprising a second nucleotide sequence consistingof 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions2 and 14 from the 5′ end of the first nucleotide sequence; and wherein2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17 from the 5′end of the first nucleotide sequence. In some embodiments, the firstnucleotide sequence consists of 19 nucleotides. In some embodiments,2′-O-methyl nucleotides are at positions 18 and 19 from the 5′ end ofthe first nucleotide sequence. In some embodiments, the secondnucleotide sequence consists of 21 nucleotides. In some embodiments,2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of thesecond nucleotide sequence.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising (a) a sense strand comprising a first nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are atpositions 3, 7-9, 12 and 17 from the 5′ end of the first nucleotidesequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2,4-6, 10, 11, and 13-16 from the 5′ end of the first nucleotide sequence;and (b) an antisense strand comprising a second nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein the nucleotides in thesecond nucleotide sequence are arranged in an alternating 1:3modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotideand 3 nucleotides are 2′-O-methyl nucleotides. In some embodiments, thefirst nucleotide sequence consists of 19 nucleotides. In someembodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the5′ end of the first nucleotide sequence. In some embodiments, the secondnucleotide sequence consists of 21 nucleotides. In some embodiments,2′-O-methyl nucleotides are at positions 19-21 from the 5′ end of thesecond nucleotide sequence. In some embodiments, the alternating 1:3modification pattern occurs 2-5 times. In some embodiments, at least twoof the alternating 1:3 modification pattern occur consecutively. In someembodiments, at least two of the alternating 1:3 modification patternoccurs nonconsecutively. In some embodiments, at least 1, 2, 3, 4, or 5alternating 1:3 modification pattern begins at nucleotide position 2, 6,10, 14, and/or 18 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 2 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 6 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 10 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 14 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 18 from the 5′ end of the antisense strand.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising (a) a sense strand comprising a first nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are atpositions 5 and 7-9 from the 5′ end of the first nucleotide sequence,and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17from the 5′ end of the first nucleotide sequence; and (b) an antisensestrand comprising a second nucleotide sequence consisting of 17 to 23nucleotides, wherein the nucleotides in the second nucleotide sequenceare arranged in an alternating 1:3 modification pattern, and wherein 1nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methylnucleotides. In some embodiments, the first nucleotide sequence consistsof 19 nucleotides. In some embodiments, 2′-O-methyl nucleotides are atpositions 18 and 19 from the 5′ end of the first nucleotide sequence. Insome embodiments, the second nucleotide sequence consists of 21nucleotides. In some embodiments, 2′-O-methyl nucleotides are atpositions 19-21 from the 5′ end of the second nucleotide sequence. Insome embodiments, the alternating 1:3 modification pattern occurs 2-5times. In some embodiments, at least two of the alternating 1:3modification pattern occur consecutively. In some embodiments, at leasttwo of the alternating 1:3 modification pattern occurs nonconsecutively.In some embodiments, at least 1, 2, 3, 4, or 5 alternating 1:3modification pattern begins at nucleotide position 2, 6, 10, 14, and/or18 from the 5′ end of the antisense strand. In some embodiments, atleast one alternating 1:3 modification pattern begins at nucleotideposition 2 from the 5′ end of the antisense strand. In some embodiments,at least one alternating 1:3 modification pattern begins at nucleotideposition 6 from the 5′ end of the antisense strand. In some embodiments,at least one alternating 1:3 modification pattern begins at nucleotideposition 10 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 14 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 18 from the 5′ end of the antisense strand.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising (a) a sense strand comprising a first nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are atpositions 5 and 7-9 from the 5′ end of the first nucleotide sequence,and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17from the 5′ end of the first nucleotide sequence; and (b) an antisensestrand comprising a second nucleotide sequence consisting of 17 to 23nucleotides, wherein the nucleotides in the second nucleotide sequenceare arranged in an alternating 1:2 modification pattern, and wherein 1nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methylnucleotides. In some embodiments, the first nucleotide sequence consistsof 19 nucleotides. In some embodiments, 2′-O-methyl nucleotides are atpositions 18 and 19 from the 5′ end of the first nucleotide sequence. Insome embodiments, the second nucleotide sequence consists of 21nucleotides. In some embodiments, 2′-O-methyl nucleotides are atpositions 18-21 from the 5′ end of the second nucleotide sequence. Insome embodiments, the alternating 1:2 modification pattern occurs 2-5times. In some embodiments, at least two of the alternating 1:2modification pattern occur consecutively. In some embodiments, at leasttwo of the alternating 1:2 modification pattern occurs nonconsecutively.In some embodiments, at least 1, 2, 3, 4, or 5 alternating 1:2modification pattern begins at nucleotide position 2, 5, 8, 14, and/or17 from the 5′ end of the antisense strand. In some embodiments, atleast one alternating 1:2 modification pattern begins at nucleotideposition 2 from the 5′ end of the antisense strand. In some embodiments,at least one alternating 1:2 modification pattern begins at nucleotideposition 5 from the 5′ end of the antisense strand. In some embodiments,at least one alternating 1:2 modification pattern begins at nucleotideposition 8 from the 5′ end of the antisense strand. In some embodiments,at least one alternating 1:2 modification pattern begins at nucleotideposition 14 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 17 from the 5′ end of the antisense strand.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising (a) a sense strand comprising a first nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are atpositions 5 and 7-9 from the 5′ end of the first nucleotide sequence,and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17from the 5′ end of the first nucleotide sequence; and (b) an antisensestrand comprising a second nucleotide sequence consisting of 17 to 23nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14,and 16 from the 5′ end of the second nucleotide sequence, and wherein2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17 fromthe 5′ end the second nucleotide sequence. In some embodiments, thefirst nucleotide sequence consists of 19 nucleotides. In someembodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the5′ end of the first nucleotide sequence. In some embodiments, the secondnucleotide sequence consists of 21 nucleotides. In some embodiments,2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of thesecond nucleotide sequence.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising: (a) a sense strand comprising a first nucleotide sequenceconsisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are atpositions 5, 9-11, and 14 from the 5′ end of the first nucleotidesequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6-8,and 12-17 from the 5′ end of the first nucleotide sequence; and (b) anantisense strand comprising a second nucleotide sequence consisting of17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2and 14 from the 5′ end of the second nucleotide sequence, and wherein2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17 from the 5′end the second nucleotide sequence. In some embodiments, the firstnucleotide sequence consists of 21 nucleotides. In some embodiments,2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of thefirst nucleotide sequence. In some embodiments, the second nucleotidesequence consists of 23 nucleotides. In some embodiments, 2′-O-methylnucleotides are at positions 18-23 from the 5′ end of the secondnucleotide sequence.

In some embodiments, any of the sense strands disclosed herein furthercomprise a TT sequence adjacent to the first nucleotide sequence.

In some embodiments, any of the sense strands disclosed herein furthercomprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore phosphorothioate internucleoside linkages. In some embodiments, atleast one phosphorothioate internucleoside linkage is between thenucleotides at positions 1 and 2 from the 5′ end of the first nucleotidesequence. In some embodiments, at least one phosphorothioateinternucleoside linkage is between the nucleotides at positions 2 and 3from the 5′ end of the first nucleotide sequence.

In some embodiments, any of the antisense strands disclosed hereinfurther comprise TT sequence adjacent to the second nucleotide sequence.In some embodiments, the antisense strand further comprises at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioateinternucleoside linkages. In some embodiments, at least onephosphorothioate internucleoside linkage is between the nucleotides atpositions 1 and 2 from the 5′ end of the second nucleotide sequence. Insome embodiments, at least one phosphorothioate internucleoside linkageis between the nucleotides at positions 2 and 3 from the 5′ end of thesecond nucleotide sequence. In some embodiments, at least onephosphorothioate internucleoside linkage is between the nucleotides atpositions 1 and 2 from the 3′ end of the second nucleotide sequence. Insome embodiments, at least one phosphorothioate internucleoside linkageis between the nucleotides at positions 2 and 3 from the 3′ end of thesecond nucleotide sequence.

In some embodiments, the first nucleotide from the 5′ end of any of thefirst nucleotide sequences disclosed herein comprises a 5′ stabilizingend cap.

In some embodiments, the first nucleotide from the 5′ end of any of thesecond nucleotide sequences disclosed herein comprise a 5′ stabilizingend cap.

In some embodiments, the first nucleotide from the 5′ end of any of thefirst nucleotide sequences disclosed herein comprises a phosphorylationblocker.

In some embodiments, the first nucleotide from the 5′ end of any of thesecond nucleotide sequences disclosed herein comprises a phosphorylationblocker.

In some embodiments, any of the first nucleotide sequences or secondnucleotide sequences disclosed herein comprise at least one modifiednucleotide selected from

R is H or alkyl (or AmNA(N-Me)) when R is alkyl);

wherein B is a nucleobase.

Disclosed herein is a short-interfering nucleic acid (siNA) moleculecomprising:

-   -   (a) a phosphorylation blocker of Formula (IV):

-   -   wherein    -   R¹ is a nucleobase,    -   R⁴ is —O—R³⁰ or —NR³¹R³²,    -   R³⁰ is C₁-C₈ substituted or unsubstituted alkyl; and    -   R³¹ and R³² together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;        and    -   (b) a short interfering nucleic acid (siNA). In some        embodiments, the siNA is any of the siNAs disclosed herein. In        some embodiments, the siNA comprises any of the sense strands,        first nucleotide sequences, antisense strands, or second        nucleotide sequences disclosed herein. In some embodiments, the        siNA comprises any of the the sense strands disclosed herein. In        some embodiments, the siNA comprises any of the antisense strand        disclosed herein. In some embodiments, the siNA comprises a        first nucleotide sequence selected from any one of SEQ ID NOs:        1-56, 103-158, and 205-260. In some embodiments, the siNA        comprises a second nucleotide sequence selected from any one of        SEQ ID NOs: 57-102, 159-204, and 261-306. In some embodiments,        the siNA comprises a sense sequence selected from any one of SEQ        ID NOs: 307-362 and 415-444. In some embodiments, the siNA        comprises an antisense sequence selected from any one of SEQ ID        NOs: 363-409, 445-533, and 536-539. In some embodiments, the        siNA comprises a ds-siNA sequence selected from any one of        ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA        further comprises any of the 5′ end caps disclosed herein. In        some embodiments, the siNA further comprises any of the        conjugated moieties disclosed herein. In some embodiments, the        siNA further comprises any of the destabilizing nucleotides        disclosed herein. In some embodiments, the siNA further        comprises any of the modified nucleotides disclosed herein.

Disclosed herein is a short-interfering nucleic acid (siNA) moleculecomprising:

-   -   (a) a 5′-stabilized end cap of Formula (Ia):

-   -   wherein    -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)—Z, or —(C₂-C₆ alkenylene)-Z andR²⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²¹R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴,    -   R²¹ and R²² are independently hydrogen or C₁-C₆ alkyl; R²¹ and        R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4; and    -   (b) a short interfering nucleic acid (siNA). In some        embodiments, the siNA comprises any of the the sense strands        disclosed herein. In some embodiments, the siNA comprises any of        the antisense strand disclosed herein. In some embodiments, the        siNA comprises a first nucleotide sequence selected from any one        of SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments,        the siNA comprises a second nucleotide sequence selected from        any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In some        embodiments, the siNA comprises a sense sequence selected from        any one of SEQ ID NOs: 307-362 and 415-444. In some embodiments,        the siNA comprises an antisense sequence selected from any one        of SEQ ID NOs: 363-409, 445-533, and 536-539. In some        embodiments, the siNA comprises a ds-siNA sequence selected from        any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the        siNA further comprises any of the phosphorylation blockers        disclosed herein. In some embodiments, the siNA further        comprises any of the conjugated moieties disclosed herein. In        some embodiments, the siNA further comprises any of the        destabilizing nucleotides disclosed herein. In some embodiments,        the siNA further comprises any of the modified nucleotides        disclosed herein.

Disclosed herein is a short-interfering nucleic acid (siNA) moleculecomprising:

-   -   (a) a 5′-stabilized end cap of Formula (Ib):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹1R²²)_(n)-Z, or —(C₂-C₆alkenylene)-Z and R³⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴,    -   R²¹ and R²² are independently hydrogen or C₁-C₆ alkyl; R²¹ and        R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4; and    -   (b) a short interfering nucleic acid (siNA). In some        embodiments, the siNA comprises any of the the sense strands        disclosed herein. In some embodiments, the siNA comprises any of        the antisense strand disclosed herein. In some embodiments, the        siNA comprises a first nucleotide sequence selected from any one        of SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments,        the siNA comprises a second nucleotide sequence selected from        any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In some        embodiments, the siNA comprises a sense sequence selected from        any one of SEQ ID NOs: 307-362 and 415-444. In some embodiments,        the siNA comprises an antisense sequence selected from any one        of SEQ ID NOs: 363-409, 445-533, and 536-539. In some        embodiments, the siNA comprises a ds-siNA sequence selected from        any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the        siNA further comprises any of the phosphorylation blockers        disclosed herein. In some embodiments, the siNA further        comprises any of the conjugated moieties disclosed herein. In        some embodiments, the siNA further comprises any of the        destabilizing nucleotides disclosed herein. In some embodiments,        the siNA further comprises any of the modified nucleotides        disclosed herein.

Disclosed herein is a short-interfering nucleic acid (siNA) moleculecomprising: (a) a 5′-stabilized end cap selected from the groupconsisting of Formula (1) to Formula (15), Formula (9X) to Formula(12X), and Formula (9Y) to Formula (12Y):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H; and (b) a shortinterfering nucleic acid (siNA). In some embodiments, the siNA comprisesany of the the sense strands disclosed herein. In some embodiments, thesiNA comprises any of the antisense strand disclosed herein. In someembodiments, the siNA comprises a first nucleotide sequence selectedfrom any one of SEQ ID NOs: 1-56, 103-158, and 205-260. In someembodiments, the siNA comprises a second nucleotide sequence selectedfrom any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In someembodiments, the siNA comprises a sense sequence selected from any oneof SEQ ID NOs: 307-362 and 415-444. In some embodiments, the siNAcomprises an antisense sequence selected from any one of SEQ ID NOs:363-409, 445-533, and 536-539. In some embodiments, the siNA comprises ads-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178.In some embodiments, the siNA further comprises any of thephosphorylation blockers disclosed herein. In some embodiments, the siNAfurther comprises any of the conjugated moieties disclosed herein. Insome embodiments, the siNA further comprises any of the destabilizingnucleotides disclosed herein. In some embodiments, the siNA furthercomprises any of the modified nucleotides disclosed herein.

Disclosed herein is a short-interfering nucleic acid (siNA) moleculecomprising: (a) a 5′-stabilized end cap selected from the groupconsisting of Formulas (1A)-(15A), Formulas (9B)-(12B), Formulas(9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas(9BY)-(12BY):

and (b) a short interfering nucleic acid (siNA). In some embodiments,the siNA comprises any of the the sense strands disclosed herein. Insome embodiments, the siNA comprises any of the antisense stranddisclosed herein. In some embodiments, the siNA comprises a firstnucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158,and 205-260. In some embodiments, the siNA comprises a second nucleotidesequence selected from any one of SEQ ID NOs: 57-102, 159-204, and261-306. In some embodiments, the siNA comprises a sense sequenceselected from any one of SEQ ID NOs: 307-362 and 415-444. In someembodiments, the siNA comprises an antisense sequence selected from anyone of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments,the siNA comprises a ds-siNA sequence selected from any one ofds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA furthercomprises any of the phosphorylation blockers disclosed herein. In someembodiments, the siNA further comprises any of the conjugated moietiesdisclosed herein. In some embodiments, the siNA further comprises any ofthe destabilizing nucleotides disclosed herein. In some embodiments, thesiNA further comprises any of the modified nucleotides disclosed herein.

Disclosed herein is a short-interfering nucleic acid (siNA) moleculecomprising:

-   -   (a) a 5′-stabilized end cap of Formula (Ic):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,

R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)-Z, or —(C₂-C₆ alkenylene)-Z andR³⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²¹R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(OOH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴,        or —NR²³SO₂R²⁴;    -   R²¹ and R²² either are independently hydrogen or C₁-C₆ alkyl, or        R²¹ and R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4; and    -   (b) a short interfering nucleic acid (siNA). In some        embodiments, the siNA comprises any of the the sense strands        disclosed herein. In some embodiments, the siNA comprises any of        the antisense strand disclosed herein. In some embodiments, the        siNA comprises a first nucleotide sequence selected from any one        of SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments,        the siNA comprises a second nucleotide sequence selected from        any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In some        embodiments, the siNA comprises a sense sequence selected from        any one of SEQ ID NOs: 307-362 and 415-444. In some embodiments,        the siNA comprises an antisense sequence selected from any one        of SEQ ID NOs: 363-409, 445-533, and 536-539. In some        embodiments, the siNA comprises a ds-siNA sequence selected from        any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the        siNA further comprises any of the phosphorylation blockers        disclosed herein. In some embodiments, the siNA further        comprises any of the conjugated moieties disclosed herein. In        some embodiments, the siNA further comprises any of the        destabilizing nucleotides disclosed herein. In some embodiments,        the siNA further comprises any of the modified nucleotides        disclosed herein.

Disclosed herein is a short-interfering nucleic acid (siNA) moleculecomprising: (a) a 5′-stabilized end cap selected from the groupconsisting of Formula (21) to Formula (35):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H; and (b) a shortinterfering nucleic acid (siNA). In some embodiments, the siNA comprisesany of the the sense strands disclosed herein. In some embodiments, thesiNA comprises any of the antisense strand disclosed herein. In someembodiments, the siNA comprises a first nucleotide sequence selectedfrom any one of SEQ ID NOs: 1-56, 103-158, and 205-260. In someembodiments, the siNA comprises a second nucleotide sequence selectedfrom any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In someembodiments, the siNA comprises a sense sequence selected from any oneof SEQ ID NOs: 307-362 and 415-444. In some embodiments, the siNAcomprises an antisense sequence selected from any one of SEQ ID NOs:363-409, 445-533, and 536-539. In some embodiments, the siNA comprises ads-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178.In some embodiments, the siNA further comprises any of thephosphorylation blockers disclosed herein. In some embodiments, the siNAfurther comprises any of the conjugated moieties disclosed herein. Insome embodiments, the siNA further comprises any of the destabilizingnucleotides disclosed herein. In some embodiments, the siNA furthercomprises any of the modified nucleotides disclosed herein.

Disclosed herein is a short-interfering nucleic acid (siNA) moleculecomprising: (a) a 5′-stabilized end cap selected from the groupconsisting of Formulas (21A)-(35A), Formulas (29B)-(32B), Formulas(29AX)-(32AX), Formulas (29AY)-(32AY), Formulas (29BX)-(32BX), andFormulas (29BY)-(32BY):

and (b) a short interfering nucleic acid (siNA). In some embodiments,the siNA comprises any of the the sense strands disclosed herein. Insome embodiments, the siNA comprises any of the antisense stranddisclosed herein. In some embodiments, the siNA comprises a firstnucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158,and 205-260. In some embodiments, the siNA comprises a second nucleotidesequence selected from any one of SEQ ID NOs: 57-102, 159-204, and261-306. In some embodiments, the siNA comprises a sense sequenceselected from any one of SEQ ID NOs: 307-362 and 415-444. In someembodiments, the siNA comprises an antisense sequence selected from anyone of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments,the siNA comprises a ds-siNA sequence selected from any one ofds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA furthercomprises any of the phosphorylation blockers disclosed herein. In someembodiments, the siNA further comprises any of the conjugated moietiesdisclosed herein. In some embodiments, the siNA further comprises any ofthe destabilizing nucleotides disclosed herein. In some embodiments, thesiNA further comprises any of the modified nucleotides disclosed herein.

Disclosed herein is a short interfering nucleic acid (siNA) moleculecomprising: (a) a sense strand comprising a first nucleotide sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%identical to an RNA corresponding to a target gene, wherein the firstnucleotide sequence comprises a nucleotide sequence of any one SEQ IDNOs: 1-56, 103-158, and 205-260; and (b) an antisense strand comprisinga second nucleotide sequence that is at least about 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding tothe target gene, wherein the second nucleotide sequence comprises anucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and261-306. In some embodiments, the siNA further comprises any of the 5′end caps disclosed herein. In some embodiments, the siNA furthercomprises any of the phosphorylation blockers disclosed herein. In someembodiments, the siNA further comprises any of the conjugated moietiesdisclosed herein. In some embodiments, the siNA further comprises any ofthe destabilizing nucleotides disclosed herein. In some embodiments, thesiNA further comprises any of the modified nucleotides disclosed herein.

Disclosed herein is a interfering nucleic acid (siNA) moleculecomprising: (a) a sense strand comprising a nucleotide sequence of anyone of SEQ ID NOs: 307-362 and 415-444; and (b) an antisense strandcomprising a nucleotide sequence of any one of SEQ ID NOs: 363-409,445-533, and 536-539.

In some embodiments, any of the siNA disclosed herein further comprise aphosphorylation blocker.

In some embodiments, the phosphorylation blocker has the structure ofFormula (IV):

wherein

-   -   R¹ is a nucleobase,    -   R⁴ is —O—R³⁰ or —NR³¹R³², R³⁰ is C₁-C₈ substituted or        unsubstituted alkyl; and    -   R³¹ and R³² together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring.

In some embodiments, R⁴ is —OCH₃ or —N(CH₂CH₂)₂O.

In some embodiments, the phosphorylation blocker is attached to the 5′end of the sense strand.

In some embodiments, the phosphorylation blocker is attached to the 5′end of the sense strand via one or more linkers independently selectedfrom a phosphodiester linker, phosphorothioate linker, andphosphorodithioate linker.

In some embodiments, the phosphorylation blocker is attached to the 3′end of the sense strand.

In some embodiments, the phosphorylation blocker is attached to the 3′end of the sense strand via one or more linkers independently selectedfrom a phosphodiester linker, phosphorothioate linker, andphosphorodithioate linker.

In some embodiments, the phosphorylation blocker is attached to the 5′end of the antisense strand. In some embodiments, the phosphorylationblocker is attached to the 5′ end of the antisense strand via one ormore linkers independently selected from a phosphodiester linker,phosphorothioate linker, and phosphorodithioate linker. In someembodiments, the phosphorylation blocker is attached to the 3′ end ofthe antisense strand. In some embodiments, the phosphorylation blockeris attached to the 3′ end of the antisense strand via one or morelinkers independently selected from a phosphodiester linker,phosphorothioate linker, and phosphorodithioate linker.

In some embodiments, any of the siNAs disclosed herein further comprisea conjugated moiety. In some embodiments, the conjugated moietycomprises a galactosamine. In some embodiments, the galactosamine isN-acetylgalactosamine (GalNAc) of Formula (VII):

wherein each n is independently 1 or 2. In some embodiments, thegalactosamine is N-acetylgalactosamine (GalNAc) of Formula (VI):

wherein

-   -   m is 1, 2, 3, 4, or 5;    -   each n is independently 1 or 2;    -   p is 0 or 1;    -   each R is independently H;    -   each Y is independently selected from —O—P(═O)(SH)—,        —O—P(═O)(O))—, —O—P(═O)(OH)—, and —O—P(S)S—;    -   Z is H or a second protecting group;    -   either L is a linker or L and Y in combination are a linker; and    -   A is H, OH, a third protecting group, an activated group, or an        oligonucleotide. In some embodiments, wherein A is an        oligonucleotide. In some embodiments, A is 1-2 oligonucleotides.        In some embodiments, the oligonucleotide is dTdT. In some        embodiments, the galactosamine is attached to the 3′ end of the        sense strand. In some embodiments, the galactosamine is attached        to the 3′ end of the sense strand via one or more linkers        independently selected from a phosphodiester linker,        phosphorothioate linker, or phosphorodithioate linker. In some        embodiments, the galactosamine is attached to the 5′ end of the        sense strand. In some embodiments, the galactosamine is attached        to the 5′ end of the sense strand via one or more linkers        independently selected from a phosphodiester linker,        phosphorothioate linker, or phosphorodithioate linker. In some        embodiments, the galactosamine is attached to the 3′ end of the        antisense strand. In some embodiments, the galactosamine is        attached to the 3′ end of the atnisense strand via one or more        linkers independently selected from a phosphodiester linker,        phosphorothioate linker, or phosphorodithioate linker. In some        embodiments, the galactosamine is attached to the 5′ end of the        antisense strand. In some embodiments, the galactosamine is        attached to the 5′ end of the atnisense strand via one or more        linkers independently selected from a phosphodiester linker,        phosphorothioate linker, or phosphorodithioate linker.

In some embodiments, any of the siNAs disclosed herein further comprisea 5′-stabilized end cap. In some embodiments, the 5′-stabilized end capis a 5′ vinyl phosphonate or deuterated 5′ vinyl phosphonate. In someembodiments, the 5′-stabilized end cap has the structure of Formula(Ia):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)—Z, or —(C₂-C₆ alkenylene)-Z andR²⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴,        or —NR²³SO₂R²⁴;    -   R²¹ and R²² either are independently hydrogen or C₁-C₆ alkyl, or        R²¹ and R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4. In some embodiments, the 5′-stabilized end        cap has the structure of    -   Formula (Ib):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴,        or —NR²³SO₂R²⁴;    -   R²¹ and R²² either are independently hydrogen or C₁-C₆ alkyl, or        R²¹ and R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4. In some embodiments, the 5′-stabilized end        cap has the structure of    -   Formula (Ic):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁴, —NR²³R²⁴,        or —NR²³SO₂R²⁴;    -   R²¹ and R²² either are independently hydrogen or C₁-C₆ alkyl, or        R²¹ and R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4. In some embodiments, R¹ is an aryl. In some        embodiments, the aryl is a phenyl. In some embodiments, the        5′-stabilized end cap is selected from the group consisting of        Formula (1) to Formula (15), Formula (9X) to Formula (12X), and        Formula (9Y) to Formula (12Y):

wherein R¹ independently is a nucleobase, aryl, heteroaryl, or H. Insome embodiments, the 5′-stabilized end cap is selected from the groupconsisting of Formulas (1A)-(15A), Formulas (9B)-(12B), Formulas(9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas(9BY)-(12BY):

In some embodiments, the 5′-stabilized end cap is selected from thegroup consisting of Formula (21) to Formula (35):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H. In some embodiments,the 5′-stabilized end cap is selected from the group consisting ofFormulas (21A)-(35A), Formulas (29B)-(32B), Formulas (29AX)-(32AX),Formulas (29AY)-(32AY), Formulas (29BX)-(32BX), and Formulas(29BY)-(32BY):

In some embodiments, the 5′-stabilized end cap is attached to the 5′ endof the antisense strand. In some embodiments, the 5′-stabilized end capis attached to the 5′ end of the antisense strand via one or morelinkers independently selected from a phosphodiester linker,phosphorothioate linker, or phosphorodithioate linker. In someembodiments, the 5′-stabilized end cap is attached to the 5′ end of thesense strand. In some embodiments, the 5′-stabilized end cap is attachedto the 5′ end of the sense strand via one or more linkers independentlyselected from a phosphodiester linker, phosphorothioate linker, orphosphorodithioate linker.

In some embodiments, any of the siNAs, sense strands, first nucleotidesequences, antisense strands, or second nucleotide sequences disclosedherein further comprise at least one thermally destabilizingnucleotides. In some embodiments, any of the antisense strands disclosedherein further comprise at least one thermally destabilizing nucleotideselected from:

In some embodiments, any of the sense strands disclosed herein compriseat least one thermally destabilizing nucleotide selected from:

In some embodiments, any of the first nucleotide sequences disclosedherein further comprise at least one thermally destabilizing nucleotideselected from:

In some embodiments, any of the second nucleotide sequences disclosedherein further comprise at least one thermally destabilizing nucleotideselected from:

In some embodiments, any of the modified nucleotides disclosed herein isa thermally destabilizing nucleotide.

In some embodiments, any of the siNAs disclosed herein specificallydownregulate or reduce expression of a target gene. In some embodiments,the target gene is a viral gene. In some embodiments, the viral gene isfrom a DNA virus. In some embodiments, the DNA virus is adouble-stranded DNA (dsDNA) virus. In some embodiments, the dsDNA virusis a hepadnavirus. In some embodiments, the hepadnavirus is a hepatitisB virus (HBV). In some embodiments, the HBV is selected from HBVgenotypes A-J. In some embodiments, the target gene is selected from theS gene or X gene of the HBV.

In some embodiments, the second nucleotide sequence is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to30 nucleotides within positions 200-720 or 1100-1700 of SEQ ID NO: 410.In some embodiments, the second nucleotide sequence is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to30 nucleotides within positions 200-280, 300-445, 460-510, 650-720,1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410. In someembodiments, the second nucleotide sequence is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30nucleotides within positions 200-230, 250-280, 300-330, 370-400,405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610of SEQ ID NO: 410. In some embodiments, the second nucleotide sequenceis at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to 15 to 30 nucleotides starting at position 203, 206,254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182,1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ IDNO: 410.

In some embodiments, the first nucleotide sequence is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30nucleotides within positions 200-720 or 1100-1700 of SEQ ID NO: 410. Insome embodiments, the first nucleotide sequence is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30nucleotides within positions 200-280, 300-445, 460-510, 650-720,1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410. In someembodiments, the first nucleotide sequence is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 nucleotideswithin positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500,670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO:410. In some embodiments, the first nucleotide sequence is at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to30 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412,415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526,1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO: 410.

In some embodiments, the first nucleotide sequence comprises anucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.

In some embodiments, the second nucleotide sequence comprises anucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and261-306.

In some embodiments, the sense strand comprises a nucleotide sequence ofany one of SEQ ID NOs: 307-362 and 415-444.

In some embodiments, the antisense strand comprises a nucleotidesequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.

In some embodiments, at least one end of the siNA is a blunt end.

In some embodiments, at least one end of the siNA comprises an overhang,wherein the overhang comprises at least one nucleotide.

In some embodiments, both ends of the siNA comprise an overhang, whereinthe overhang comprises at least one nucleotide.

In some embodiments, the siNA is selected from ds-siNA-001 tods-siNA-0178.

In some embodiments, at least one 2′-fluoro nucleotide or 2′-O-methylnucleotide is a 2′-fluoro or 2-O-methyl nucleotide mimic of Formula (V):

wherein

-   -   R¹ is independently a nucleobase, aryl, heteroaryl, or H, Q¹ and        Q² are independently S or O,    -   R⁵ is independently —OCD₃, —F, or —OCH₃, and    -   R⁶ and R⁷ are independently H, D, or CD3.

In some embodiments, the 2′-fluoro or 2′-O-methyl nucleotide mimic is anucleotide mimic of Formula (16)-Formula (20):

wherein R¹ is a nucleobase and R² is independently F or —OCH₃.

Further disclosed herein are compositions comprising any of the siNAsdisclosed herein. In some embodiments, the siNA targets an S gene ofHBV. In some embodiments, the siNA specifically downregulates or reducesexpression of the S gene of HBV. In some embodiments, the siNA targetsan X gene of HBV. In some embodiments, the siNA specificallydownregulates or reduces expression of the X gene of HBV. In someembodiments, the siNA comprises a first nucleotide sequence. In someembodiments, the first nucleotide sequence comprises a nucleotidesequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260. In someembodiments, the siNA comprises a second nucleotide sequence. In someembodiments, the second nucleotide sequence comprises a nucleotidesequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In someembodiments, the siNA comprises a sense strand. In some embodiments, thesense strand comprises a nucleotide sequence of any one of SEQ ID NOs:307-362 and 415-444. In some embodiments, the siNA comprises anantisense strand. In some embodiments, the antisense strand comprises anucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and536-539. In some embodiments, the siNA further comprises any of the 5′end caps disclosed herein. In some embodiments, the siNA furthercomprises any of the phosphorylation blockers disclosed herein. In someembodiments, the siNA further comprises any of the conjugated moietiesdisclosed herein. In some embodiments, the siNA further comprises any ofthe destabilized nucleotides disclosed herein. In some embodiments, thesiNA further comprises any of the modified nucleotides disclosed herein.

Further disclosed herein are compositions comprising 2, 3, 4, 5, 6, 7,8, 9, 10 or more of any of the siNAs disclosed herein. In someembodiments, at least 1, 2, 3, 4, 5, or more siNAs target an S gene ofHBV. In some embodiments, at least 1, 2, 3, 4, 5, or more siNAsspecifically downregulate or reduce expression of the S gene of HBV. Insome embodiments, at least 1, 2, 3, 4, 5, or more siNAs target an X geneof HBV. In some embodiments, at least 1, 2, 3, 4, 5, or more siNAsspecifically downregulate or reduce expression of the X gene of HBV. Insome embodiments, the siNA comprises a first nucleotide sequence. Insome embodiments, the first nucleotide sequence comprises a nucleotidesequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260. In someembodiments, the siNA comprises a second nucleotide sequence. In someembodiments, the second nucleotide sequence comprises a nucleotidesequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In someembodiments, the siNA comprises a sense strand. In some embodiments, thesense strand comprises a nucleotide sequence of any one of SEQ ID NOs:307-362 and 415-444. In some embodiments, the siNA comprises anantisense strand. In some embodiments, the antisense strand comprises anucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and536-539. In some embodiments, the siNA further comprises any of the 5′end caps disclosed herein. In some embodiments, the siNA furthercomprises any of the phosphorylation blockers disclosed herein. In someembodiments, the siNA further comprises any of the conjugated moietiesdisclosed herein. In some embodiments, the siNA further comprises any ofthe destabilized nucleotides disclosed herein. In some embodiments, thesiNA further comprises any of the modified nucleotides disclosed herein.

In some embodiments, any of the compositions disclosed herein furthercomprise an additional HBV treatment agent. In some embodiments, theadditional HBV treatment agent is selected from a nucleotide analog,nucleoside analog, a capsid assembly modulator (CAM), a recombinantinterferon, an entry inhibitor, a small molecule immunomodulator andoligonucleotide therapy. In some embodiments, the oligonucleotidetherapy is an additional siNA. In some embodiments, the additional siNAis selected from any of ds-siNA-001 to ds-siNA-0178. In someembodiments, the oligonucleotide therapy is an antisense oligonucleotide(ASO), NAPs, or STOPs. In some embodiments, the ASO is ASO 1 or ASO 2.In some embodiments, the ASO specifically targets the S gene of HBV. Insome embodiments, the ASO specifically targets the X gene of HBV. Insome embodiments, the additional HBV treatment agent is selected fromHBV STOPS™ ALG-010133, HBV CAM ALG-000184, ASO 1, recombinant interferonalpha 2b, IFN-α, PEG-IFN-α-2a, lamivudine, telbivudine, adefovirdipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovirdisoproxil, NVR3-778, BAY41-4109, JNJ-632, JNJ-3989 (ARO-HBV), RG6004,GSK3228836, REP-2139, REP-2165, AB-729, VIR-2218, RG6346 (DCR-HBVS),JNJ-6379, GLS4, ABI-HO731, JNJ-440, NZ-4, RG7907, EDP-514, AB-423,AB-506, ABI-H03733 and ABI-H2158.

In some embodiments, any of the compositions disclosed herein furthercomprise a liver disease treatment agent. In some embodiments, the liverdisease treatment agent is selected from a peroxisomeproliferator-activator receptor (PPAR) agonist, farnesoid X receptor(FXR) agonist, lipid-altering agent, and incretin-based therapy. In someembodiments, the PPAR agonist is selected from a PPARα agonist, dualPPARα/δ agonist, PPARγ agonist, and dual PPARα/γ agonist. In someembodiments, the dual PPARα agonist is a fibrate. In some embodiments,the PPARα/δ agonist is elafibranor. In some embodiments, the PPARγagonist is a thiazolidinedione (TZD). In some embodiments, TZD ispioglitazone. In some embodiments, the dual PPARα/γ agonist issaroglitazar. In some embodiments, the FXR agonist is obeticholic acis(OCA). In some embodiments, the lipid-altering agent is aramchol. Insome embodiments, the incretin-based therapy is a glucagon-like peptide1 (GLP-1) receptor agonist or dipeptidyl peptidase 4 (DPP-4) inhibitor.In some embodiments, the GLP-1 receptor agonist is exenatide orliraglutide. In some embodiments, the DPP-4 inhibitor is sitagliptin orvildapliptin.

Further disclosed herein are methods of treating a disease in a subjectin need thereof, comprising administering to the subject any of thesiNAs disclosed herein. In some embodiments, the siNA comprises a firstnucleotide sequence. In some embodiments, the first nucleotide sequencecomprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158,and 205-260. In some embodiments, the siNA comprises a second nucleotidesequence. In some embodiments, the second nucleotide sequence comprisesa nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and261-306. In some embodiments, the siNA comprises a sense strand. In someembodiments, the sense strand comprises a nucleotide sequence of any oneof SEQ ID NOs: 307-362 and 415-444. In some embodiments, the siNAcomprises an antisense strand. In some embodiments, the antisense strandcomprises a nucleotide sequence of any one of SEQ ID NOs: 363-409,445-533, and 536-539. In some embodiments, the siNA further comprisesany of the 5′ end caps disclosed herein. In some embodiments, the siNAfurther comprises any of the phosphorylation blockers disclosed herein.In some embodiments, the siNA further comprises any of the conjugatedmoieties disclosed herein. In some embodiments, the siNA furthercomprises any of the destabilized nucleotides disclosed herein. In someembodiments, the siNA further comprises any of the modified nucleotidesdisclosed herein.

Further disclosed herein are methods of treating a disease in a subjectin need thereof, comprising administering to the subject any of thecompositions disclosed herein. In some embodiments, the compositioncomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of any of thesiNAs disclosed herein. In some embodiments, the siNA comprises a firstnucleotide sequence. In some embodiments, the first nucleotide sequencecomprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158,and 205-260. In some embodiments, the siNA comprises a second nucleotidesequence. In some embodiments, the second nucleotide sequence comprisesa nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and261-306. In some embodiments, the siNA comprises a sense strand. In someembodiments, the sense strand comprises a nucleotide sequence of any oneof SEQ ID NOs: 307-362 and 415-444. In some embodiments, the siNAcomprises an antisense strand. In some embodiments, the antisense strandcomprises a nucleotide sequence of any one of SEQ ID NOs: 363-409,445-533, and 536-539. In some embodiments, the siNA further comprisesany of the 5′ end caps disclosed herein. In some embodiments, the siNAfurther comprises any of the phosphorylation blockers disclosed herein.In some embodiments, the siNA further comprises any of the conjugatedmoieties disclosed herein. In some embodiments, the siNA furthercomprises any of the destabilized nucleotides disclosed herein. In someembodiments, the siNA further comprises any of the modified nucleotidesdisclosed herein. In some embodiments, the composition further comprisesany of the additional HBV treatment agents disclosed herein. In someembodiments, the disease is a viral disease. In some embodiments, theviral disease is caused by a DNA virus. In some embodiments, the DNAvirus is a double stranded DNA (dsDNA) virus. In some embodiments, thedsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus isa hepatitis B virus (HBV). In some embodiments, the HBV is selected fromHBV genotypes A-J. In some embodiments, the method further comprisesadministering an additional HBV treatment agent. In some embodiments,the siNA or the composition and the additional HBV treatment agent areadministered concurrently. In some embodiments, the siNA or thecomposition and the additional HBV treatment agent are administeredsequentially. In some embodiments, the siNA or the composition isadministered prior to administering the additional HBV treatment agent.In some embodiments, the siNA or the composition is administered afteradministering the additional HBV treatment agent. In some embodiments,the additional HBV treatment agent is selected from a nucleotide analog,nucleoside analog, a capsid assembly modulator (CAM), a recombinantinterferon, an entry inhibitor, a small molecule immunomodulator andoligonucleotide therapy. In some embodiments, the oligonucleotidetherapy is an additional siNA. In some embodiments, the additional siNAis selected from any of ds-siNA-001 to ds-siNA-0178. In someembodiments, the oligonucleotide therapy is an antisense oligonucleotide(ASO), NAPs, or STOPs. In some embodiments, the ASO is ASO 1 or ASO 2.In some embodiments, the additional HBV treatment agent is selected fromHBV STOPS' ALG-010133, HBV CAM ALG-000184, ASO 1, recombinant interferonalpha 2b, IFN-α, PEG-IFN-α-2a, lamivudine, telbivudine, adefovirdipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovirdisoproxil, NVR3-778, BAY41-4109, JNJ-632, JNJ-3989 (ARO-HBV), RG6004,GSK3228836, REP-2139, REP-2165, AB-729, VIR-2218, RG6346 (DCR-HBVS),JNJ-6379, GLS4, ABI-HO731, JNJ-440, NZ-4, RG7907, EDP-514, AB-423,AB-506, ABI-H03733 and ABI-H2158.

In some embodiments, the disease is a liver disease. In someembodiments, the liver disease is a nonalcoholic fatty liver disease(NAFLD) or hepatocellular carcinoma (HCC). In some embodiments, theNAFLD is nonalcoholic steatohepatitis (NASH). In some embodiments, themethod further comprises administering to the subject a liver diseasetreatment agent. In some embodiments, the liver disease treatment agentis selected from a peroxisome proliferator-activator receptor (PPAR)agonist, farnesoid X receptor (FXR) agonist, lipid-altering agent, andincretin-based therapy. In some embodiments, the PPAR agonist isselected from a PPARα agonist, dual PPARα/δ agonist, PPARγ agonist, anddual PPARα/γ agonist. In some embodiments, the dual PPARα agonist is afibrate. In some embodiments, the PPARα/δ agonist is elafibranor. Insome embodiments, the PPARγ agonist is a thiazolidinedione (TZD). Insome embodiments, TZD is pioglitazone. In some embodiments, the dualPPARα/γ agonist is saroglitazar. In some embodiments, the FXR agonist isobeticholic acis (OCA). In some embodiments, the lipid-altering agent isaramchol. In some embodiments, the incretin-based therapy is aglucagon-like peptide 1 (GLP-1) receptor agonist or dipeptidyl peptidase4 (DPP-4) inhibitor. In some embodiments, the GLP-1 receptor agonist isexenatide or liraglutide. In some embodiments, the DPP-4 inhibitor issitagliptin or vildapliptin. In some embodiments, the siNA orcomposition and the liver disease treatment agent are administeredconcurrently. In some embodiments, the siNA or composition and the liverdisease treatment agent are administered sequentially. In someembodiments, the siNA or composition is administered prior toadministering the liver disease treatment agent. In some embodiments,the siNA or composition is administered after administering the liverdisease treatment agent.

In some embodiments, the siNA or the composition is administered at adose of at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg 14mg/kg, or 15 mg/kg. In some embodiments, the siNA or the composition isadministered at a dose of between 0.5 mg/kg to 50 mg/kg, 0.5 mg/kg to 40mg/kg 0.5 mg/kg to 30 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 40 mg/kg, 1mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 3 mg/kg to 50 mg/kg, 3 mg/kg to40 mg/kg, 3 mg/kg to 30 mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to 15 mg/kg,3 mg/kg to 10 mg/kg, 4 mg/kg to 50 mg/kg, 4 mg/kg to 40 mg/kg, 4 mg/kgto 30 mg/kg, 4 mg/kg to 20 mg/kg, 4 mg/kg to 15 mg/kg, 4 mg/kg to 10mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg to 40 mg/kg, 5 mg/kg to 30 mg/kg, 5mg/kg to 20 mg/kg, 5 mg/kg to 15 mg/kg, or 5 mg/kg to 10 mg/kg.

In some embodiments, the siNA or the composition is administered atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, thesiNA or the composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 times a day, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times aweek, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a month. Insome embodiments, the siNA or the composition are administered at leastonce every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or 21 days. In some embodiments, the siNA or the compositionis administered for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 52, 53, 54, or 55weeks.

In some embodiments, the siNA or the composition is administered at asingle dose of 5 mg/kg. In some embodiments, the siNA or the compositionis administered at a single dose of 10 mg/kg. In some embodiments, thesiNA or the composition is administered at three doses of 10 mg/kg oncea week. In some embodiments, the siNA or the composition is administeredat three doses of 10 mg/kg once every three days. In some embodiments,the siNA or the composition is administered at five doses of 10 mg/kgonce every three days. In some embodiments, the siNA or the compositionis administered at six doses of ranging from 1 mg/kg to 15 mg/kg, 1mg/kg to 10 mg/kg, 2 mg/kg to 15 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to15 mg/kg, or 3 mg/kg to 10 mg/kg. In some embodiments, the first doseand second dose are administered at least 3 days apart. In someembodiments, the second dose and third dose are administered at least 4days apart. In some embodiments, the third dose and fourth dose, fourthdose and fifth dose, or fifth dose and sixth dose are administered atleast 7 days apart.

In some embodiments, any of the siNAs or the compositions disclosedherein are formulated as a particle or viral vector. In someembodiments, the siNA or the composition are administered in a particleor viral vector. In some embodiments, the viral vector is a vector ofadenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpessimplex virus, lentivirus, measles virus, picornavirus, poxvirus,retrovirus, or rhabdovirus. In some embodiments, the viral vector is arecombinant viral vector. In some embodiments, the viral vector isselected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. Insome embodiments, the siNA or the composition is administeredsystemically. In some embodiments, the siNA or the composition isadministered locally. In some embodiments, the siNA or the compositionis administered intravenously, subcutaneously, or intramuscularly.

In some embodiments, any of the siRNAs or compositions disclosed hereinare used in the manufacture of a medicament for treating a disease. Insome embodiments, the disease is a viral disease. In some embodiments,the viral disease is caused by a DNA virus. In some embodiments, the DNAvirus is a double stranded DNA (dsDNA virus). In some embodiments, thedsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus isa hepatitis B virus (HBV). In some embodiments, the HBV is selected fromHBV genotypes A-J. In some embodiments, an additional HBV treatmentagent is further used in the manufacture of the medicament. In someembodiments, the additional HBV treatment agent is selected from anucleotide analog, nucleoside analog, a capsid assembly modulator (CAM),a recombinant interferon, an entry inhibitor, a small moleculeimmunomodulator and oligonucleotide therapy. In some embodiments, theoligonucleotide therapy is an additional siNA. In some embodiments, theadditional siNA is selected from any of ds-siNA-001 to ds-siNA-0178. Insome embodiments, the oligonucleotide therapy is an antisenseoligonucleotide (ASO), NAPs, or STOPs. In some embodiments, the ASO isASO 1 or ASO 2. In some embodiments, the additional HBV treatment agentis selected from HBV STOPS™ ALG-010133, HBV CAM ALG-000184, ASO 1,recombinant interferon alpha 2b, IFN-α, PEG-IFN-α-2a, lamivudine,telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofoviralafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109, JNJ-632,JNJ-3989 (ARO-HBV), RG6004, GSK3228836, REP-2139, REP-2165, AB-729,VIR-2218, RG6346 (DCR-HBVS), JNJ-6379, GLS4, ABI-HO731, JNJ-440, NZ-4,RG7907, EDP-514, AB-423, AB-506, ABI-H03733 and ABI-H2158.

In some embodiments, any of the siRNAs or compositions disclosed hereinare used in the manufacture of a medicament for treating a disease. Insome embodiments, the disease is a liver disease. In some embodiments,the liver disease is a nonalcoholic fatty liver disease (NAFLD) orhepatocellular carcinoma (HCC). In some embodiments, the NAFLD isnonalcoholic steatohepatitis (NASH). In some embodiments, the siNAcomprises a first nucleotide sequence. In some embodiments, the firstnucleotide sequence comprises a nucleotide sequence of any one SEQ IDNOs: 1-56, 103-158, and 205-260. In some embodiments, the siNA comprisesa second nucleotide sequence. In some embodiments, the second nucleotidesequence comprises a nucleotide sequence of any one of SEQ ID NOs:57-102, 159-204, and 261-306. In some embodiments, the siNA comprises asense strand. In some embodiments, the sense strand comprises anucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444. Insome embodiments, the siNA comprises an antisense strand. In someembodiments, the antisense strand comprises a nucleotide sequence of anyone of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments,the siNA further comprises any of the 5′ end caps disclosed herein. Insome embodiments, the siNA further comprises any of the phosphorylationblockers disclosed herein. In some embodiments, the siNA furthercomprises any of the conjugated moieties disclosed herein. In someembodiments, the siNA further comprises any of the destabilizednucleotides disclosed herein. In some embodiments, the siNA furthercomprises any of the modified nucleotides disclosed herein. In someembodiments, a liver disease treatment agent is further used in themanufacture of the medicament. In some embodiments, the liver diseasetreatment agent is selected from a peroxisome proliferator-activatorreceptor (PPAR) agonist, farnesoid X receptor (FXR) agonist,lipid-altering agent, and incretin-based therapy. In some embodiments,the PPAR agonist is selected from a PPARα agonist, dual PPARα/δ agonist,PPARγ agonist, and dual PPARα/γ agonist. In some embodiments, the dualPPARα agonist is a fibrate. In some embodiments, the PPARα/S agonist iselafibranor. In some embodiments, the PPARγ agonist is athiazolidinedione (TZD). In some embodiments, TZD is pioglitazone. Insome embodiments, the dual PPARα/γ agonist is saroglitazar. In someembodiments, the FXR agonist is obeticholic acis (OCA). In someembodiments, the lipid-altering agent is aramchol. In some embodiments,the incretin-based therapy is a glucagon-like peptide 1 (GLP-1) receptoragonist or dipeptidyl peptidase 4 (DPP-4) inhibitor. In someembodiments, the GLP-1 receptor agonist is exenatide or liraglutide. Insome embodiments, the DPP-4 inhibitor is sitagliptin or vildapliptin.

In some embodiments, any of the siNAs disclosed herein is used as amedicament. In some embodiments, the siNA comprises a first nucleotidesequence. In some embodiments, the first nucleotide sequence comprises anucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.In some embodiments, the siNA comprises a second nucleotide sequence. Insome embodiments, the second nucleotide sequence comprises a nucleotidesequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In someembodiments, the siNA comprises a sense strand. In some embodiments, thesense strand comprises a nucleotide sequence of any one of SEQ ID NOs:307-362 and 415-444. In some embodiments, the siNA comprises anantisense strand. In some embodiments, the antisense strand comprises anucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and536-539. In some embodiments, the siNA further comprises any of the 5′end caps disclosed herein. In some embodiments, the siNA furthercomprises any of the phosphorylation blockers disclosed herein. In someembodiments, the siNA further comprises any of the conjugated moietiesdisclosed herein. In some embodiments, the siNA further comprises any ofthe destabilized nucleotides disclosed herein. In some embodiments, thesiNA further comprises any of the modified nucleotides disclosed herein.

In some embodiments, any of the compositions disclosed herein are usedas a medicament. In some embodiments, the composition comprises any ofthe siNAs disclosed herein. In some embodiments, the siNA comprises afirst nucleotide sequence. In some embodiments, the first nucleotidesequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56,103-158, and 205-260. In some embodiments, the siNA comprises a secondnucleotide sequence. In some embodiments, the second nucleotide sequencecomprises a nucleotide sequence of any one of SEQ ID NOs: 57-102,159-204, and 261-306. In some embodiments, the siNA comprises a sensestrand. In some embodiments, the sense strand comprises a nucleotidesequence of any one of SEQ ID NOs: 307-362 and 415-444. In someembodiments, the siNA comprises an antisense strand. In someembodiments, the antisense strand comprises a nucleotide sequence of anyone of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments,the siNA further comprises any of the 5′ end caps disclosed herein. Insome embodiments, the siNA further comprises any of the phosphorylationblockers disclosed herein. In some embodiments, the siNA furthercomprises any of the conjugated moieties disclosed herein. In someembodiments, the siNA further comprises any of the destabilizednucleotides disclosed herein. In some embodiments, the siNA furthercomprises any of the modified nucleotides disclosed herein.

In some embodiments, any of the siNAs disclosed herein are used in thetreatment of a disease. In some embodiments, the siNA comprises a firstnucleotide sequence. In some embodiments, the first nucleotide sequencecomprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158,and 205-260. In some embodiments, the siNA comprises a second nucleotidesequence. In some embodiments, the second nucleotide sequence comprisesa nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and261-306. In some embodiments, the siNA comprises a sense strand. In someembodiments, the sense strand comprises a nucleotide sequence of any oneof SEQ ID NOs: 307-362 and 415-444. In some embodiments, the siNAcomprises an antisense strand. In some embodiments, the antisense strandcomprises a nucleotide sequence of any one of SEQ ID NOs: 363-409,445-533, and 536-539. In some embodiments, the siNA further comprisesany of the 5′ end caps disclosed herein. In some embodiments, the siNAfurther comprises any of the phosphorylation blockers disclosed herein.In some embodiments, the siNA further comprises any of the conjugatedmoieties disclosed herein. In some embodiments, the siNA furthercomprises any of the destabilized nucleotides disclosed herein. In someembodiments, the siNA further comprises any of the modified nucleotidesdisclosed herein. In some embodiments, the disease is a viral disease.In some embodiments, the viral disease is caused by a DNA virus. In someembodiments, the DNA virus is a double stranded DNA (dsDNA virus). Insome embodiments, the dsDNA virus is a hepadnavirus. In someembodiments, the hepadnavirus is a hepatitis B virus (HBV). In someembodiments, the HBV is selected from HBV genotypes A-J. In someembodiments, the disease is a liver disease. In some embodiments, theliver disease is a nonalcoholic fatty liver disease (NAFLD) orhepatocellular carcinoma (HCC). In some embodiments, the NAFLD isnonalcoholic steatohepatitis (NASH).

In some embodiments, any of the compositions disclosed herein are usedin the treatment of a disease. In some embodiments, the compositioncomprises any of the siNAs disclosed herein. In some embodiments, thesiNA comprises a first nucleotide sequence. In some embodiments, thefirst nucleotide sequence comprises a nucleotide sequence of any one SEQID NOs: 1-56, 103-158, and 205-260. In some embodiments, the siNAcomprises a second nucleotide sequence. In some embodiments, the secondnucleotide sequence comprises a nucleotide sequence of any one of SEQ IDNOs: 57-102, 159-204, and 261-306. In some embodiments, the siNAcomprises a sense strand. In some embodiments, the sense strandcomprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and415-444. In some embodiments, the siNA comprises an antisense strand. Insome embodiments, the antisense strand comprises a nucleotide sequenceof any one of SEQ ID NOs: 363-409, 445-533, and 536-539. In someembodiments, the siNA further comprises any of the 5′ end caps disclosedherein. In some embodiments, the siNA further comprises any of thephosphorylation blockers disclosed herein. In some embodiments, the siNAfurther comprises any of the conjugated moieties disclosed herein. Insome embodiments, the siNA further comprises any of the destabilizednucleotides disclosed herein. In some embodiments, the siNA furthercomprises any of the modified nucleotides disclosed herein. In someembodiments, the disease is a viral disease. In some embodiments, theviral disease is caused by a DNA virus. In some embodiments, the DNAvirus is a double stranded DNA (dsDNA virus). In some embodiments, thedsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus isa hepatitis B virus (HBV). In some embodiments, the HBV is selected fromHBV genotypes A-J. In some embodiments, the disease is a liver disease.In some embodiments, the liver disease is a nonalcoholic fatty liverdisease (NAFLD) or hepatocellular carcinoma (HCC). In some embodiments,the NAFLD is nonalcoholic steatohepatitis (NASH).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary siNA molecule.

FIG. 2 illustrates an exemplary siNA molecule.

FIGS. 3A-3G illustrate exemplary double-stranded siNA molecules.

FIG. 4 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with ds-siNA-0160, ds-siNA-0165, ds-siNA-0163, or ds-siNA-0166.

FIG. 5A shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0160 (G03).

FIG. 5B shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0160 (G15).

FIG. 5C shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0160 (G03).

FIG. 5D shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G03), or ds-siNA-0109 (G09).

FIGS. 5E-5F show a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0169 (G18).

FIG. 5G shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0169 (G04).

FIG. 5H shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0169 (G04).

FIG. 5I shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0169 (G04), or ds-siNA-0147 (G08).

FIG. 5J shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0166 (G06), or ds-siNA-0153 (G14).

FIG. 5K shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0163 (G05), or ds-siNA-0119 (G13).

FIG. 6A shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G15), or ds-siNA-080 (G14).

FIG. 6B shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0169 (G16), or ds-siNA-081 (G13).

FIG. 7A shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0165 (G18), or ds-siNA-0127 (G17).

FIG. 7B shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0168 (G20), or ds-siNA-0150 (G19).

FIG. 8A shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G06), ASO 1 (G18), or acombination of ds-siNA-0160 and ASO 1 (G20).

FIG. 8B shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G06), ASO 1 (G18), or acombination of ds-siNA-0160 and ASO 1 (G20).

FIG. 8C shows a graph of a synergy analysis of a combination therapywith unconjugated forms of ds-siNA-0164 and ASO 2 (e.g., ds-siNA-0160and ASO 1 without GalNac).

FIG. 9 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0166 (G03), ds-siNA-0155 (G08), ords-siNA-0157.

FIG. 10 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0165 (G10), ds-siNA-0160 (G06), or acombination therapy with ds-siNA-0160 and ds-siNA-0165 (G14).

FIG. 11 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0165 (G05), or ds-siNA-0144 (G11).

FIG. 12 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0163 (G04), ds-siNA-0122 (G09), ords-siNA-0123 (G13).

FIG. 13 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G 15) or ds-siNA-0147 (G 19).

FIG. 14 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G 15, square), ds-siNA-0109 (G 21, circle), ords-siNA-0172 (G 27, diamond).

FIG. 15 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G 01, circle), ds-siNA-0109 (G 07, square),ds-siNA-0119 (G 11, triangle), or ds-siNA-0153 (G 13, diamond).

FIG. 16 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G 01, circle), ASO 1 (G 20, square), ds-siNA-0147(G 24, diamond), or a combination of ASO 1 and ds-siNA-0147 (G 25,triangle).

FIG. 17 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G 01, circle), ASO 1 (G 20, square), ds-siNA-0109(G 26, diamond), or a combination of ASO 1 and ds-siNA-0109 (G 27,triangle).

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are short interfering nucleic acid (siNA) moleculescomprising modified nucleotides. The siNA molecules described herein maybe double-stranded siNA (ds-siNA) molecules. The siNA moleculesdescribed herein may comprise modified nucleotides selected from2′-O-methyl nucleotides and 2′-fluoro nucleotides. The siNA moleculesdescribed herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morephosphorothioate internucleoside linkages. The siNA molecules describedherein may comprise a phosphorylation blocker. The siNA moleculesdescribed herein may comprise a 5′-stabilized end cap. The siNAmolecules described herein may comprise a galactosamine. The siNAmolecules described herein may comprise one or more blunt ends. The siNAmolecules described herein may comprise one or more overhangs.

Further disclosed herein are short interfering nucleic acid (siNA)molecules comprising (a) a phosphorylation blocker; and (b) a shortinterfering nucleic acid (siNA). The siNA may comprise at least 5nucleotides. The nucleotides may be modified nucleotides, non-modifiednucleotides, or any combination thereof. The nucleotides may beribonucleotides, deoxyribonucleotides, or any combination thereof. ThesiNA may be single-stranded. Alternatively, the siNA is double-stranded.The double-stranded siNA may comprise one or more blunt ends. Thedouble-stranded siNA may comprise one or more overhangs. Thedouble-stranded siNA may comprise a blunt end and an overhang.

Further disclosed herein are short interfering nucleic acid (siNA)molecules comprising (a) a conjugated moiety; and (b) a shortinterfering nucleic acid (siNA). The siNA may comprise at least 5nucleotides. The nucleotides may be modified nucleotides, non-modifiednucleotides, or any combination thereof. The nucleotides may beribonucleotides, deoxyribonucleotides, or any combination thereof. ThesiNA may be single-stranded. Alternatively, the siNA is double-stranded.The double-stranded siNA may comprise one or more blunt ends. Thedouble-stranded siNA may comprise one or more overhangs. Thedouble-stranded siNA may comprise a blunt end and an overhang.

Further disclosed herein are short interfering nucleic acid (siNA)molecules comprising (a) a 5′-stabilized end cap; and (b) a shortinterfering nucleic acid (siNA). The siNA may comprise at least 5nucleotides. The nucleotides may be modified nucleotides, non-modifiednucleotides, or any combination thereof. The nucleotides may beribonucleotides, deoxyribonucleotides, or any combination thereof. ThesiNA may be single-stranded. Alternatively, the siNA is double-stranded.The double-stranded siNA may comprise one or more blunt ends. Thedouble-stranded siNA may comprise one or more overhangs. Thedouble-stranded siNA may comprise a blunt end and an overhang.

Further disclosed herein are short interfering nucleic acid (siNA)molecules comprising (a) at least one phosphorylation blocker,conjugated moiety, or 5′-stabilized end cap; and (b) a short interferingnucleic acid (siNA). The siNA may comprise at least 5 nucleotides. Thenucleotides may be modified nucleotides, non-modified nucleotides, orany combination thereof. The nucleotides may be ribonucleotides,deoxyribonucleotides, or any combination thereof. The siNA may besingle-stranded. Alternatively, the siNA is double-stranded. Thedouble-stranded siNA may comprise one or more blunt ends. Thedouble-stranded siNA may comprise one or more overhangs. Thedouble-stranded siNA may comprise a blunt end and an overhang.

An exemplary siNA molecule of the present disclosure is shown in FIG. 1. As shown in FIG. 1 , an exemplary siNA molecule comprises a sensestrand (101) and an antisense strand (102). The sense strand (101) maycomprise a first oligonucleotide sequence (103). The firstoligonucleotide sequence (103) may comprise one or more phosphorothioateinternucleoside linkages (109). The phosphorothioate internucleosidelinkage (109) may be between the nucleotides at the 5′ or 3′ terminalend of the first oligonucleotide sequence (103). The phosphorothioateinternucleoside linkage (109) may be between the first three nucleotidesfrom the 5′ end of the first oligonucleotide sequence (103). The firstoligonucleotide sequence (103) may comprise one or more 2′-fluoronucleotides (110). The first oligonucleotide sequence (103) may compriseone or more 2′-O-methyl nucleotides (111). The first oligonucleotidesequence (103) may comprise 15 or more modified nucleotidesindependently selected from 2′-fluoro nucleotides (110) and 2′-O-methylnucleotides (111). The sense strand (101) may further comprise aphosphorylation blocker (105). The sense strand (101) may furthercomprise a galactosamine (106). The antisense strand (102) may comprisea second oligonucleotide sequence (104). The second oligonucleotidesequence (104) may comprise one or more phophorothioate internucleosidelinkages (109). The phosphorothioate internucleoside linkage (109) maybe between the nucleotides at the 5′ or 3′ terminal end of the secondoligonucleotide sequence (104). The phosphorothioate internucleosidelinkage (109) may be between the first three nucleotides from the 5′ endof the second oligonucleotide sequence (104). The phosphorothioateinternucleoside linkage (109) may be between the first three nucleotidesfrom the 3′ end of the second oligonucleotide sequence (104). The secondoligonucleotide sequence (104) may comprise one or more 2′-fluoronucleotides (110). The second oligonucleotide sequence (104) maycomprise one or more 2′-O-methyl nucleotides (111). The secondoligonucleotide sequence (104) may comprise 15 or more modifiednucleotides independently selected from 2′-fluoro nucleotides (110) and2′-O-methyl nucleotides (111). The antisense strand (102) may furthercomprise a 5′-stabilized end cap (107). The siNA may further compriseone or more blunt ends. Alternatively, or additionally, one end of thesiNA may comprise an overhang (108). The overhang (108) may be part ofthe sense strand (101). The overhang (108) may be part of the antisensestrand (102). The overhang (108) may be distinct from the firstnucleotide sequence (103). The overhang (108) may be distinct from thesecond nucleotide sequence (104). The overhang (108) may be part of thefirst nucleotide sequence (103). The overhang (108) may be part of thesecond nucleotide sequence (104). The overhang (108) may comprise 1 ormore nucleotides. The overhang (108) may comprise 1 or moredeoxyribonucleotides. The overhang (108) may comprise 1 or more modifiednucleotides. The overhang (108) may comprise 1 or more modifiedribonucleotides. The sense strand (101) may be shorter than theantisense strand (102). The sense strand (101) may be the same length asthe antisense strand (102). The sense strand (101) may be longer thanthe antisense strand (102).

An exemplary siNA molecule of the present disclosure is shown in FIG. 2. As shown in FIG. 2 , an exemplary siNA molecule comprises a sensestrand (201) and an antisense strand (202). The sense strand (201) maycomprise a first oligonucleotide sequence (203). The firstoligonucleotide sequence (203) may comprise one or more phophorothioateinternucleoside linkages (209). The phosphorothioate internucleosidelinkage (209) may be between the nucleotides at the 5′ or 3′ terminalend of the first oligonucleotide sequence (203). The phosphorothioateinternucleoside linkage (209) may be between the first three nucleotidesfrom the 5′ end of the first oligonucleotide sequence (203). The firstoligonucleotide sequence (203) may comprise one or more 2′-fluoronucleotides (210). The first oligonucleotide sequence (203) may compriseone or more 2′-O-methyl nucleotides (211). The first oligonucleotidesequence (203) may comprise 15 or more modified nucleotidesindependently selected from 2′-fluoro nucleotides (210) and 2′-O-methylnucleotides (211). The sense strand (201) may further comprise aphosphorylation blocker (205). The sense strand (201) may furthercomprise a galactosamine (206). The antisense strand (202) may comprisea second oligonucleotide sequence (204). The second oligonucleotidesequence (204) may comprise one or more phophorothioate internucleosidelinkages (209). The phosphorothioate internucleoside linkage (209) maybe between the nucleotides at the 5′ or 3′ terminal end of the secondoligonucleotide sequence (204). The phosphorothioate internucleosidelinkage (209) may be between the first three nucleotides from the 5′ endof the second oligonucleotide sequence (204). The phosphorothioateinternucleoside linkage (209) may be between the first three nucleotidesfrom the 3′ end of the second oligonucleotide sequence (204). The secondoligonucleotide sequence (204) may comprise one or more 2′-fluoronucleotides (210). The second oligonucleotide sequence (204) maycomprise one or more 2′-O-methyl nucleotides (211). The secondoligonucleotide sequence (204) may comprise 15 or more modifiednucleotides independently selected from 2′-fluoro nucleotides (210) and2′-O-methyl nucleotides (211). The antisense strand (202) may furthercomprise a 5′-stabilized end cap (207). The siNA may further compriseone or more overhangs (208). The overhang (208) may be part of the sensestrand (201). The overhang (208) may be part of the antisense strand.(202). The overhang (208) may be distinct from the first nucleotidesequence (203). The overhang (208) may be distinct from the secondnucleotide sequence (204). The overhang (208) may be part of the firstnucleotide sequence (203). The overhang (208) may be part of the secondnucleotide sequence (204). The overhang (208) may be adjacent to the 3′end of the first nucleotide sequence (203). The overhang (208) may beadjacent to the 5′ end of the first nucleotide sequence (203). Theoverhang (208) may be adjacent to the 3′ end of the second nucleotidesequence (204). The overhang (208) may be adjacent to the 5′ end of thesecond nucleotide sequence (204). The overhang (208) may comprise 1 ormore nucleotides. The overhang (208) may comprise 1 or moredeoxyribonucleotides. The overhang (208) may comprise a TT sequence. Theoverhang (208) may comprise 1 or more modified nucleotides. The overhang(208) may comprise 1 or more modified nucleotides disclosed herein(e.g., 2-fluoro nucleotide, 2′-O-methyl nucleotide, 2′-fluoro nucleotidemimic, 2′-O-methyl nucleotide mimic, or a nucleotide comprising amodified nucleobase). The overhang (208) may comprise 1 or more modifiedribonucleotides. The sense strand (201) may be shorter than theantisense strand (202). The sense strand (201) may be the same length asthe antisense strand (202). The sense strand (201) may be longer thanthe antisense strand (202).

FIGS. 3A-3G depict exemplary ds-siNA modification patterns. As shown inFIGS. 3A-3G, an exemplary ds-siNA molecule may have the followingformula:

5′-A_(n) ¹B_(n) ²A_(n) ³B_(n) ⁴A_(n) ⁵B_(n) ⁶A_(n) ⁷B_(n) ⁸A_(n) ⁹-3′3′-C_(q) ¹A_(q) ²B_(q) ³A_(q) ⁴B_(q) ⁵A_(q) ⁶B_(q) ⁷A_(q) ⁸B_(q) ⁹A_(q)¹⁰B_(q) ¹¹A_(q) ¹²-5′wherein:

-   -   the top strand is a sense strand comprising a first nucleotide        sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,        90%, 95%, or 100% identical to an RNA corresponding to a target        gene, wherein the first nucleotide sequence comprises 15 to 30        nucleotides;    -   the bottom strand is an antisense strand comprising a second        nucleotide sequence that is at least about 60%, 65%, 70%, 75%,        80%, 85%, 90%, 95%, or 100% complementary to the RNA        corresponding to the target gene, wherein the second nucleotide        sequence comprises 15 to 30 nucleotides;    -   each A is independently a 2′-O-methyl nucleotide or a nucleotide        comprising a 5′ stabilized end cap or phosphorylation blocker;    -   B is a 2′-fluoro nucleotide;    -   C represents overhanging nucleotides and is a 2′-O-methyl        nucleotide;    -   n¹=1-4 nucleotides in length;    -   each n², n⁶, n⁸, q³, q⁵, q⁷, q⁹, q¹¹, and q¹² is independently        0-1 nucleotides in length;    -   each n³ and n⁴ is independently 1-3 nucleotides in length;    -   n⁵ is 1-10 nucleotides in length;    -   n⁷ is 0-4 nucleotides in length;    -   each n⁹, q¹, and q² is independently 0-2 nucleotides in length;    -   q⁴ is 0-3 nucleotides in length;    -   q⁶ is 0-5 nucleotides in length;    -   q⁸ is 2-7 nucleotides in length; and    -   q¹⁰ is 2-11 nucleotides in length.

The ds-siNA may further comprise a conjugated moiety. The conjugatedmoiety may comprise any of the galactosamines disclosed herein. Theds-siNA may further comprise (i) phosphorothioate internucleosidelinkages between the nucleotides at positions 1 and 2 and positions 2and 3 from the 5′ end of the sense strand; and (ii) phosphorothioateinternucleoside linkages between the nucleotides at positions 1 and 2;positions 2 and 3; positions 19 and 20; and positions 20 and 21 from the5′ end of the antisense strand. The ds-siNA may further comprise a5′-stabilizing end cap. The 5′-stabilizing end cap may be a vinylphosphonate. The 5′-stabilizing end cap may be attached to the 5′ end ofthe antisense strand. In some embodiments, the 2′-O-methyl nucleotide atposition 1 from the 5′ end of the sense strand is further modified tocontain a 5′ stabilizing end cap. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 5′ end of the antisense strand isfurther modified to contain a 5′ stabilizing end cap. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is further modified to contain a phosphorylationblocker. In some embodiments, the 2′-O-methyl nucleotide at position 1from the 3′ end of the sense strand is further modified to contain aphosphorylation blocker. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 5′ end of the antisense strand is furthermodified to contain a phosphorylation blocker. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 3′ end of the antisensestrand is further modified to contain a phosphorylation blocker. Anexemplary ds-siNA molecule may have the following formula:

5′-A₂₋₄B₁A₁₋₃B₂₋₃A₂₋₁₀B₀₋₁A₀₋₄B₀₋₁A₀₋₂-3′3′-C₂A₀₋₂B₀₋₁A₀₋₃B₀₋₁A₀₋₅B₀₋₁A₂₋₇B₁A₂₋₁₁B₁A₁-5′wherein:

-   -   the top strand is a sense strand comprising a first nucleotide        sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,        90%, 95%, or 100% identical to an RNA corresponding to a target        gene, wherein the first nucleotide sequence comprises 15 to 30        nucleotides;    -   the bottom strand is an antisense strand comprising a second        nucleotide sequence that is at least about 60%, 65%, 70%, 75%,        80%, 85%, 90%, 95%, or 100% complementary to the RNA        corresponding to the target gene, wherein the second nucleotide        sequence comprises 15 to 30 nucleotides;    -   each A is independently a 2′-O-methyl nucleotide or a nucleotide        comprising a 5′ stabilized end cap or phosphorylation blocker;    -   B is a 2′-fluoro nucleotide;    -   C represents overhanging nucleotides and is a 2′-O-methyl        nucleotide.        The ds-siNA may further comprise a conjugated moiety. The        conjugated moiety may comprise any of the galactosamines        disclosed herein. The ds-siNA may further comprise (i)        phosphorothioate internucleoside linkages between the        nucleotides at positions 1 and 2 and positions 2 and 3 from the        5′ end of the sense strand; and (ii) phosphorothioate        internucleoside linkages between the nucleotides at positions 1        and 2; positions 2 and 3; positions 19 and 20; and positions 20        and 21 from the 5′ end of the antisense strand. The ds-siNA may        further comprise a 5′-stabilizing end cap. The 5′-stabilizing        end cap may be a vinyl phosphonate. The vinyl phosphonate may be        a deuterated vinyl phosphonate. The deuterated vinyl phosphonate        may be a mono-deuterated vinyl phosphonate. The deuterated vinyl        phosphonate may be a mono-di-deuterated vinyl phosphonate. The        5′-stabilizing end cap may be attached to the 5′ end of the        antisense strand. The 5′-stabilizing end cap may be attached to        the 3′ end of the antisense strand. The 5′-stabilizing end cap        may be attached to the 5′ end of the sense strand. The        5′-stabilizing end cap may be attached to the 3′ end of the        sense strand. In some embodiments, the 2′-O-methyl nucleotide at        position 1 from the 5′ end of the sense strand is further        modified to contain a 5′ stabilizing end cap. In some        embodiments, the 2′-O-methyl nucleotide at position 1 from the        5′ end of the antisense strand is further modified to contain a        5′ stabilizing end cap. In some embodiments, the 2′-O-methyl        nucleotide at position 1 from the 5′ end of the sense strand is        further modified to contain a phosphorylation blocker. In some        embodiments, the 2′-O-methyl nucleotide at position 1 from the        3′ end of the sense strand is further modified to contain a        phosphorylation blocker. In some embodiments, the 2′-O-methyl        nucleotide at position 1 from the 5′ end of the antisense strand        is further modified to contain a phosphorylation blocker. In        some embodiments, the 2′-O-methyl nucleotide at position 1 from        the 3′ end of the antisense strand is further modified to        contain a phosphorylation blocker.

The exemplary ds-siNA shown in FIGS. 3A-3G comprise (i) a sense strandcomprising 19-21 nucleotides; and (ii) an antisense strand comprising21-23 nucleotides. The ds-siNA may further comprise (iii) a conjugatedmoiety, wherein the conjugated moiety is attached to the 3′ end of theantisense strand. The ds-siNA may comprise a 2 nucleotide overhangconsisting of nucleotides at positions 20 and 21 from the 5′ end of theantisense strand. The ds-siNA may comprise a 2 nucleotide overhangconsisting of nucleotides at positions 22 and 23 from the 5′ end of theantisense strand. The ds-siNA may further comprise 1, 2, 3, 4, 5, 6 ormore phosphorothioate (ps) internucleoside linkages. At least onephosphorothioate internucleoside linkage may be between the nucleotidesat positions 1 and 2 or positions 2 and 3 from the 5′ end of the sensestrand. At least one phosphorothioate internucleoside linkage may bebetween the nucleotides at positions 1 and 2 or positions 2 and 3 fromthe 5′ end of the antisense strand. At least one phosphorothioateinternucleoside linkage may be between the nucleotides at positions 19and 20, positions 20 and 21, positions 21 and 22, or positions 22 and 23from the 5′ end of the antisense strand. As shown in FIGS. 3A-3G, 4-6nucleotides in the sense strand may be 2′-fluoro nucleotides. As shownin FIGS. 3A-3G, 2-5 nucleotides in the antisense strand may be 2′-fluoronucleotides. As shown in FIGS. 3A-3G, 13-15 nucleotides in the sensestrand may be 2′-O-methyl nucleotides. As shown in FIGS. 3A-3G, 14-19nucleotides in the antisense strand may be 2′-O-methyl nucleotides. Asshown in FIGS. 3A-3G, the ds-siNA does not contain a base pair between2′-fluoro nucleotides on the sense and antisense strands. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is further modified to contain a 5′ stabilizing endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 fromthe 5′ end of the antisense strand is further modified to contain a 5′stabilizing end cap. In some embodiments, the 2′-O-methyl nucleotide atposition 1 from the 5′ end of the sense strand is further modified tocontain a phosphorylation blocker. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 3′ end of the sense strand is furthermodified to contain a phosphorylation blocker. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 5′ end of the antisensestrand is further modified to contain a phosphorylation blocker. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end ofthe antisense strand is further modified to contain a phosphorylationblocker.

As shown in FIG. 3A, a ds-siNA may comprise (a) a sense strandconsisting of 19 nucleotides, wherein 2′-fluoro nucleotides are atpositions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, andwherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11,13-16, 18, and 19 from the 5′ end of the sense strand; (b) an antisensestrand consisting of 21 nucleotides, wherein nucleotides at positions 2and 14 from the 5′ end of the antisense strand are 2′-fluoronucleotides; and wherein nucleotides at positions 1, 3-13, and 15-21 are2′-O-methyl nucleotides. The ds-siNA may further comprise a conjugatedmoiety attached to the 3′ end of the sense strand. The ds-siNA mayfurther comprise (i) phosphorothioate internucleoside linkages betweenthe nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′end of the sense strand; and (ii) phosphorothioate internucleosidelinkages between the nucleotides at positions 1 and 2; positions 2 and3; positions 19 and 20; and positions 20 and 21 from the 5′ end of theantisense strand. In some embodiments, the 2′-O-methyl nucleotide atposition 1 from the 5′ end of the sense strand is further modified tocontain a 5′ stabilizing end cap. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 5′ end of the antisense strand isfurther modified to contain a 5′ stabilizing end cap. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is further modified to contain a phosphorylationblocker. In some embodiments, the 2′-O-methyl nucleotide at position 1from the 3′ end of the sense strand is further modified to contain aphosphorylation blocker. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 5′ end of the antisense strand is furthermodified to contain a phosphorylation blocker. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 3′ end of the antisensestrand is further modified to contain a phosphorylation blocker. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is a d2vd3 nucleotide. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 5′ end of the antisensestrand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 3′ end of the sense strand is a d2vd3nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position1 from the 3′ end of the antisense strand is a d2vd3 nucleotide. In someembodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on thesense strand or antisense strand is a 2′-fluoro nucleotide mimic. Insome embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides onthe sense strand or antisense strand is a f4P, f2P, or fX nucleotide. Insome embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide onthe sense or antisense strand is a 2′-O-methyl nucleotide mimic.

As shown in FIG. 3B, a ds-siNA may comprise (a) a sense strandconsisting of 19 nucleotides, wherein 2′-fluoro nucleotides are atpositions 3, 7, 8, and 17 from the 5′ end of the sense strand, andwherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 9-16, 18,and 19 from the 5′ end of the sense strand; (b) an antisense strandconsisting of 21 nucleotides, wherein nucleotides at positions 2 and 14from the 5′ end of the antisense strand are 2′-fluoro nucleotides; andwherein nucleotides at positions 1, 3-13, and 15-21 are 2′-O-methylnucleotides. The ds-siNA may further comprise a conjugated moietyattached to the 3′ end of the sense strand. The ds-siNA may furthercomprise (i) phosphorothioate internucleoside linkages between thenucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ endof the sense strand; and (ii) phosphorothioate internucleoside linkagesbetween the nucleotides at positions 1 and 2; positions 2 and 3;positions 19 and 20; and positions 20 and 21 from the 5′ end of theantisense strand. In some embodiments, the 2′-O-methyl nucleotide atposition 1 from the 5′ end of the sense strand is further modified tocontain a 5′ stabilizing end cap. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 5′ end of the antisense strand isfurther modified to contain a 5′ stabilizing end cap. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is further modified to contain a phosphorylationblocker. In some embodiments, the 2′-O-methyl nucleotide at position 1from the 3′ end of the sense strand is further modified to contain aphosphorylation blocker. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 5′ end of the antisense strand is furthermodified to contain a phosphorylation blocker. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 3′ end of the antisensestrand is further modified to contain a phosphorylation blocker. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is a d2vd3 nucleotide. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 5′ end of the antisensestrand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 3′ end of the sense strand is a d2vd3nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position1 from the 3′ end of the antisense strand is a d2vd3 nucleotide. In someembodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on thesense strand or antisense strand is a 2′-fluoro nucleotide mimic. Insome embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides onthe sense strand or antisense strand is a f4P, f2P, or fX nucleotide. Insome embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide onthe sense or antisense strand is a 2′-O-methyl nucleotide mimic.

As shown in FIG. 3C, a ds-siNA may comprise (a) a sense strandconsisting of 19 nucleotides, wherein 2′-fluoro nucleotides are atpositions 3, 7-9, 12 and 17 from the 5′ end of the sense strand, andwherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11,13-16, 18, and 19 from the 5′ end of the sense strand; (b) an antisensestrand consisting of 21 nucleotides, wherein the nucleotides in theantisense strand comprise an alternating 1:3 modification pattern, andwherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are2′-O-methyl nucleotides. The ds-siNA may further comprise a conjugatedmoiety attached to the 3′ end of the sense strand. The ds-siNA mayfurther comprise (i) phosphorothioate internucleoside linkages betweenthe nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′end of the sense strand; and (ii) phosphorothioate internucleosidelinkages between the nucleotides at positions 1 and 2; positions 2 and3; positions 19 and 20; and positions 20 and 21 from the 5′ end of theantisense strand. The ds-siNA may comprise 2-5 alternating 1:3modification patterns on the antisense strand. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strandis further modified to contain a 5′ stabilizing end cap. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe antisense strand is further modified to contain a 5′ stabilizing endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 fromthe 5′ end of the sense strand is further modified to contain aphosphorylation blocker. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 3′ end of the sense strand is further modified tocontain a phosphorylation blocker. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 5′ end of the antisense strand isfurther modified to contain a phosphorylation blocker. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end ofthe antisense strand is further modified to contain a phosphorylationblocker. In some embodiments, the 2′-O-methyl nucleotide at position 1from the 5′ end of the sense strand is a d2vd3 nucleotide. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe antisense strand is a d2vd3 nucleotide. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strandis a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 3′ end of the antisense strand is a d2vd3nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoronucleotides on the sense strand or antisense strand is a 2′-fluoronucleotide mimic. In some embodiments, at least 1, 2, 3, 4 or more2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fXnucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoronucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. Insome embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide onthe sense or antisense strand is a 2′-O-methyl nucleotide mimic.

As shown in FIG. 3D, a ds-siNA may comprise (a) a sense strandconsisting of 19 nucleotides, wherein 2′-fluoro nucleotides are atpositions 5 and 7-9 from the 5′ end of the sense strand, and wherein2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′end of the sense strand; (b) an antisense strand consisting of 21nucleotides, wherein the nucleotides in the antisense strand comprise analternating 1:3 modification pattern, and wherein 1 nucleotide is a2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides. Theds-siNA may further comprise a conjugated moiety attached to the 3′ endof the sense strand. The ds-siNA may further comprise (i)phosphorothioate internucleoside linkages between the nucleotides atpositions 1 and 2 and positions 2 and 3 from the 5′ end of the sensestrand; and (ii) phosphorothioate internucleoside linkages between thenucleotides at positions 1 and 2; positions 2 and 3; positions 19 and20; and positions 20 and 21 from the 5′ end of the antisense strand. Theds-siNA may comprise 2-5 alternating 1:3 modification patterns on theantisense strand. The alternating 1:3 modification pattern may start atthe nucleotide at any of positions 2, 6, 10, 14, and/or 18 from the 5′end of the antisense strand. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 5′ end of the sense strand is furthermodified to contain a 5′ stabilizing end cap. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 5′ end of the antisensestrand is further modified to contain a 5′ stabilizing end cap. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is further modified to contain a phosphorylationblocker. In some embodiments, the 2′-O-methyl nucleotide at position 1from the 3′ end of the sense strand is further modified to contain aphosphorylation blocker. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 5′ end of the antisense strand is furthermodified to contain a phosphorylation blocker. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 3′ end of the antisensestrand is further modified to contain a phosphorylation blocker. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is a d2vd3 nucleotide. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 5′ end of the antisensestrand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 3′ end of the sense strand is a d2vd3nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position1 from the 3′ end of the antisense strand is a d2vd3 nucleotide. In someembodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on thesense strand or antisense strand is a 2′-fluoro nucleotide mimic. Insome embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides onthe sense strand is a f4P, f2P, or fX nucleotide. In some embodiments,at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the antisensestrand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1,2, 3, 4 or more 2′-O-methyl nucleotide on the sense or antisense strandis a 2′-O-methyl nucleotide mimic.

As shown in FIG. 3E, a ds-siNA may comprise (a) a sense strandconsisting of 19 nucleotides, wherein 2′-fluoro nucleotides are atpositions 5 and 7-9 from the 5′ end of the sense strand, and wherein2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′end of the sense strand; (b) an antisense strand consisting of 21nucleotides, wherein the nucleotides in the antisense strand comprise analternating 1:2 modification pattern, and wherein 1 nucleotide is a2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl nucleotides. Theds-siNA may further comprise a conjugated moiety attached to the 3′ endof the sense strand. The ds-siNA may further comprise (i)phosphorothioate internucleoside linkages between the nucleotides atpositions 1 and 2 and positions 2 and 3 from the 5′ end of the sensestrand; and (ii) phosphorothioate internucleoside linkages between thenucleotides at positions 1 and 2; positions 2 and 3; positions 19 and20; and positions 20 and 21 from the 5′ end of the antisense strand. Theds-siNA may comprise 2-5 alternating 1:2 modification patterns on theantisense strand. The alternating 1:2 modification pattern may start atthe nucleotide at any of positions 2, 5, 8, 14, and/or 17 from the 5′end of the antisense strand. In some embodiments, the ds-siNA comprises(a) a sense strand consisting of 19 nucleotides, wherein 2′-fluoronucleotides are at positions 5 and 7-9 from the 5′ end of the sensestrand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and10-19 from the 5′ end of the sense strand; (b) an antisense strandconsisting of 21 nucleotides, wherein 2′-fluoro nucleotides are atpositions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand,and wherein 2′-O-methyl nucleotides are at positions 1, 3, 4, 6, 7,9-13, 15, 16, and 18-21 from the 5′ end of the sense strand. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe sense strand is further modified to contain a 5′ stabilizing endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 fromthe 5′ end of the antisense strand is further modified to contain a 5′stabilizing end cap. In some embodiments, the 2′-O-methyl nucleotide atposition 1 from the 5′ end of the sense strand is further modified tocontain a phosphorylation blocker. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 3′ end of the sense strand is furthermodified to contain a phosphorylation blocker. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 5′ end of the antisensestrand is further modified to contain a phosphorylation blocker. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end ofthe antisense strand is further modified to contain a phosphorylationblocker. In some embodiments, the 2′-O-methyl nucleotide at position 1from the 5′ end of the sense strand is a d2vd3 nucleotide. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe antisense strand is a d2vd3 nucleotide. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strandis a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 3′ end of the antisense strand is a d2vd3nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoronucleotides on the sense strand or antisense strand is a 2′-fluoronucleotide mimic. In some embodiments, at least 1, 2, 3, 4 or more2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fXnucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoronucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. Insome embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide onthe sense or antisense strand is a 2′-O-methyl nucleotide mimic.

As shown in FIG. 3F, a ds-siNA may comprise (a) a sense strandconsisting of 19 nucleotides, wherein 2′-fluoro nucleotides are atpositions 5 and 7-9 from the 5′ end of the sense strand, and wherein2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′end of the sense strand; (b) an antisense strand consisting of 21nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14,and 16 from the 5′ end of the antisense strand, and wherein 2′-O-methylnucleotides are at positions 1, 3-5, 7-13, 15, and 17-21 from the 5′ endof the antisense strand. The ds-siNA may further comprise a conjugatedmoiety attached to the 3′ end of the sense strand. The ds-siNA mayfurther comprise (i) phosphorothioate internucleoside linkages betweenthe nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′end of the sense strand; and (ii) phosphorothioate internucleosidelinkages between the nucleotides at positions 1 and 2; positions 2 and3; positions 19 and 20; and positions 20 and 21 from the 5′ end of theantisense strand. In some embodiments, at least 1, 2, 3, 4 or more2′-fluoro nucleotides on the sense strand or antisense strand is a f4P,f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more2′-fluoro nucleotides on the sense strand or antisense strand is a f4Pnucleotide. In some embodiments, at least 1, 2, 3, or 4 of the2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end ofthe antisense strand is a f4P nucleotide. In some embodiments, at leastone of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the5′ end of the antisense strand is a f4P nucleotide. In some embodiments,at least two of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16from the 5′ end of the antisense strand is a f4P nucleotide. In someembodiments, less than or equal to 3 of the 2′-fluoro-nucleotides atpositions 2, 6, 14, and 16 from the 5′ end of the antisense strand is af4P nucleotide. In some embodiments, less than or equal to 2 of the2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end ofthe antisense strand is a f4P nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 2 from the 5′ end of the antisensestrand is a f4P nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 6 from the 5′ end of the antisensestrand is a f4P nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 14 from the 5′ end of the antisensestrand is a f4P nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 16 from the 5′ end of the antisensestrand is a f4P nucleotide. In some embodiments, at least 1, 2, 3, 4 ormore 2′-fluoro nucleotides on the sense strand or antisense strand is af2P nucleotide. In some embodiments, at least 1, 2, 3, or 4 of the2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end ofthe antisense strand is a f2P nucleotide. In some embodiments, at leastone of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the5′ end of the antisense strand is a f2P nucleotide. In some embodiments,at least two of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16from the 5′ end of the antisense strand is a f2P nucleotide. In someembodiments, less than or equal to 3 of the 2′-fluoro-nucleotides atpositions 2, 6, 14, and 16 from the 5′ end of the antisense strand is af2P nucleotide. In some embodiments, less than or equal to 2 of the2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end ofthe antisense strand is a f2P nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 2 from the 5′ end of the antisensestrand is a f2P nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 6 from the 5′ end of the antisensestrand is a f2P nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 14 from the 5′ end of the antisensestrand is a f2P nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 16 from the 5′ end of the antisensestrand is a f2P nucleotide. In some embodiments, at least 1, 2, 3, 4 ormore 2′-fluoro nucleotides on the sense strand or antisense strand is afX nucleotide. In some embodiments, at least 1, 2, 3, or 4 of the2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end ofthe antisense strand is a fX nucleotide. In some embodiments, at leastone of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the5′ end of the antisense strand is a fX nucleotide. In some embodiments,at least two of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16from the 5′ end of the antisense strand is a fX nucleotide. In someembodiments, less than or equal to 3 of the 2′-fluoro-nucleotides atpositions 2, 6, 14, and 16 from the 5′ end of the antisense strand is afX nucleotide. In some embodiments, less than or equal to 2 of the2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end ofthe antisense strand is a fX nucleotide. In some embodiments, the2′-fluoro-nucleotide at position 2 from the 5′ end of the antisensestrand is a fX nucleotide. In some embodiments, the 2′-fluoro-nucleotideat position 6 from the 5′ end of the antisense strand is a fXnucleotide. In some embodiments, the 2′-fluoro-nucleotide at position 14from the 5′ end of the antisense strand is a fX nucleotide. In someembodiments, the 2′-fluoro-nucleotide at position 16 from the 5′ end ofthe antisense strand is a fX nucleotide. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strandis further modified to contain a 5′ stabilizing end cap. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe antisense strand is further modified to contain a 5′ stabilizing endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 fromthe 5′ end of the sense strand is further modified to contain aphosphorylation blocker. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 3′ end of the sense strand is further modified tocontain a phosphorylation blocker. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 5′ end of the antisense strand isfurther modified to contain a phosphorylation blocker. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end ofthe antisense strand is further modified to contain a phosphorylationblocker. In some embodiments, the 2′-O-methyl nucleotide at position 1from the 5′ end of the sense strand is a d2vd3 nucleotide. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe antisense strand is a d2vd3 nucleotide. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strandis a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 3′ end of the antisense strand is a d2vd3nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoronucleotides on the sense strand or antisense strand is a 2′-fluoronucleotide mimic. In some embodiments, at least 1, 2, 3, 4 or more2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fXnucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoronucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. Insome embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide onthe sense or antisense strand is a 2′-O-methyl nucleotide mimic.

As shown in FIG. 3G, a ds-siNA may comprise (a) a sense strandconsisting of 21 nucleotides, wherein 2′-fluoro nucleotides are atpositions 5, 9-11, 14, and 19 from the 5′ end of the sense strand, andwherein 2′-O-methyl nucleotides are at positions 1-4, 6-8, 12, 13,15-18, 20, and 21 from the 5′ end of the sense strand; and (b) anantisense strand consisting of 23 nucleotides, wherein 2′-flouronucleodies are at positions 2 and 14 from the 5′ end of the antisensestrand, and wherein 2′-O-methyl nucleotides are at positions 1, 3-13,and 15-23 from the 5′ end of the antisense strand. The ds-siNA mayfurther comprise a conjugated moiety attached to the 3′ end of the sensestrand. The ds-siNA may further comprise (i) phosphorothioateinternucleoside linkages between the nucleotides at positions 1 and 2and positions 2 and 3 from the 5′ end of the sense strand; and (ii)phosphorothioate internucleoside linkages between the nucleotides atpositions 1 and 2; positions 2 and 3; positions 19 and 20; and positions20 and 21 from the 5′ end of the antisense strand. In some embodiments,the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sensestrand is further modified to contain a 5′ stabilizing end cap. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe antisense strand is further modified to contain a 5′ stabilizing endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 fromthe 5′ end of the sense strand is further modified to contain aphosphorylation blocker. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 3′ end of the sense strand is further modified tocontain a phosphorylation blocker. In some embodiments, the 2′-O-methylnucleotide at position 1 from the 5′ end of the antisense strand isfurther modified to contain a phosphorylation blocker. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end ofthe antisense strand is further modified to contain a phosphorylationblocker. In some embodiments, the 2′-O-methyl nucleotide at position 1from the 5′ end of the sense strand is a d2vd3 nucleotide. In someembodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end ofthe antisense strand is a d2vd3 nucleotide. In some embodiments, the2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strandis a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotideat position 1 from the 3′ end of the antisense strand is a d2vd3nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoronucleotides on the sense strand or antisense strand is a 2′-fluoronucleotide mimic. In some embodiments, at least 1, 2, 3, 4 or more2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fXnucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoronucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. Insome embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide onthe sense or antisense strand is a 2′-O-methyl nucleotide mimic.

Any of the siNAs disclosed herein may comprise a sense strand and anantisense strand. The sense strand may comprise a first nucleotidesequence that is 15 to 30 nucleotides in length. The antisense strandmay comprise a second nucleotide sequence that is 15 to 30 nucleotidesin length.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andthe nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19from the 5′ end of the first nucleotide sequence is a 2′-fluoronucleotide; and (b) an antisense strand comprising a second nucleotidesequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 100% complementary to the RNA corresponding to the target gene,wherein the second nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andthe nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19from the 5′ end of the first nucleotide sequence is a 2′-fluoronucleotide; and (b) an antisense strand comprising a second nucleotidesequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 100% complementary to the RNA corresponding to the target gene,wherein the second nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andthe nucleotide at position 7 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide; and (b) an antisense strandcomprising a second nucleotide sequence that is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNAcorresponding to the target gene, wherein the second nucleotidesequence: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15or more modified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide, wherein at least one modifiednucleotide is a 2′-O-methyl nucleotide and at least one modifiednucleotide is a 2′-fluoro nucleotide.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andthe nucleotide at position 7, 9, 10, and/or 11 from the 5′ end of thefirst nucleotide sequence is a 2′-fluoro nucleotide; and (b) anantisense strand comprising a second nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to the RNA corresponding to the target gene, wherein thesecond nucleotide sequence: (i) is 15 to 30 nucleotides in length; and(ii) comprises 15 or more modified nucleotides independently selectedfrom a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein atleast one modified nucleotide is a 2′-O-methyl nucleotide and at leastone modified nucleotide is a 2′-fluoro nucleotide.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide; and (b) anantisense strand comprising a second nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to the RNA corresponding to the target gene, wherein thesecond nucleotide sequence: (i) is 15 to 30 nucleotides in length; and(ii) comprises 15 or more modified nucleotides independently selectedfrom a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein atleast one modified nucleotide is a 2′-O-methyl nucleotide and thenucleotide at position 2, 5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′end of the second nucleotide sequence is a 2′-fluoro nucleotide.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide; and (b) anantisense strand comprising a second nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to the RNA corresponding to the target gene, wherein thesecond nucleotide sequence: (i) is 15 to 30 nucleotides in length; and(ii) comprises 15 or more modified nucleotides independently selectedfrom a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein atleast one modified nucleotide is a 2′-O-methyl nucleotide and thenucleotide at position 2 of the second nucleotide sequence is a2′-fluoro nucleotide.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide; and (iii)comprises 1 or more phosphorothioate internucleoside linkage; and (b) anantisense strand comprising a second nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to the RNA corresponding to the target gene, wherein thesecond nucleotide sequence: (i) is 15 to 30 nucleotides in length; (ii)comprises 15 or more modified nucleotides independently selected from a2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least onemodified nucleotide is a 2′-O-methyl nucleotide and at least onemodified nucleotide is a 2′-fluoro nucleotide; and (iii) comprises 1 ormore phosphorothioate internucleoside linkage.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide; and (b) anantisense strand comprising a second nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to the RNA corresponding to the target gene, wherein thesecond nucleotide sequence: (i) is 15 to 30 nucleotides in length; and(ii) comprises 15 or more modified nucleotides independently selectedfrom a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein atleast one modified nucleotide is a 2′-O-methyl nucleotide and at leastone modified nucleotide is a 2′-fluoro nucleotide, wherein the ds-siNAmay further comprise a phosphorylation blocker, a galactosamine, or5′-stabilized end cap.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (I) a sense strand comprising (A) a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide; and (B) aphosphorylation blocker or a galactosamine; and (II) an antisense strandcomprising a second nucleotide sequence that is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNAcorresponding to the target gene, wherein the second nucleotidesequence: (a) is 15 to 30 nucleotides in length; and (b) comprises 15 ormore modified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide, wherein at least one modifiednucleotide is a 2′-O-methyl nucleotide and at least one modifiednucleotide is a 2′-fluoro nucleotide.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (I) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (a) is 15 to 30 nucleotides inlength; and (b) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide; and (II) anantisense strand comprising (A) a second nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to the RNA corresponding to the target gene, wherein thesecond nucleotide sequence: (i) is 15 to 30 nucleotides in length; and(ii) comprises 15 or more modified nucleotides independently selectedfrom a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein atleast one modified nucleotide is a 2′-O-methyl nucleotide and at leastone modified nucleotide is a 2′-fluoro nucleotide; and (B) a5′-stabilized end cap.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (I) a sense strand comprising (A) a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides inlength; and (ii) comprises 15 or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide,wherein at least one modified nucleotide is a 2′-O-methyl nucleotide andat least one modified nucleotide is a 2′-fluoro nucleotide; and (B) aphosphorylation blocker or a galactosamine; and (II) an antisense strandcomprising (A) a second nucleotide sequence that is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNAcorresponding to the target gene, wherein the second nucleotidesequence: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15or more modified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide, wherein at least one modifiednucleotide is a 2′-O-methyl nucleotide and at least one modifiednucleotide is a 2′-fluoro nucleotide; and (B) a 5′-stabilized end cap.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a firstnucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% identical to an RNA corresponding to a target gene,wherein the first nucleotide sequence comprises a nucleotide sequence ofany one SEQ ID NOs: 1-56, 103-158, and 205-260; and (b) an antisensestrand comprising a second nucleotide sequence that is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNAcorresponding to the target gene, wherein the second nucleotide sequencecomprises a nucleotide sequence of any one of SEQ ID NOs: 57-102,159-204, and 261-306. In some embodiments, the double-stranded shortinterfering nucleic acid (ds-siNA) molecule comprises: (a) a sensestrand comprising a first nucleotide sequence that is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 1⁰⁰% identical to an RNAcorresponding to a target gene, wherein the first nucleotide sequencecomprises a nucleotide sequence as shown in Tables 1-3; and (b) anantisense strand comprising a second nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%complementary to the RNA corresponding to the target gene, wherein thesecond nucleotide sequence comprises a nucleotide sequence as shown inTables 1-3.

In some embodiments, the double-stranded short interfering nucleic acid(ds-siNA) molecule comprises: (a) a sense strand comprising a nucleotidesequence of any one of SEQ ID NOs: 307-362 and 415-444; and (b) anantisense strand comprising a nucleotide sequence of any one of SEQ IDNOs: 363-409, 445-533, and 536-539. In some embodiments, the ds-siNAmolecule comprises a double-stranded molecule as identified by theduplex ID (e.g., ds-siNA-001 to ds-siNA-0178) shown in Tables 6 and 10.

Further disclosed herein are compositions comprising two or more of thesiNA molecules described herein.

Further disclosed herein are compositions comprising any of the siNAmolecule described and a pharmaceutically acceptable carrier or diluent.

Further disclosed herein are compositions comprising two or more of thesiNA molecules described herein for use as a medicament.

Further disclosed herein are compositions comprising any of the siNAmolecule described and a pharmaceutically acceptable carrier or diluentfor use as a medicament.

Further disclosed herein are methods of treating a disease in a subjectin need thereof, the method comprising administering to the subject anyof the siNA molecules described herein.

Further disclosed herein are uses of any of the siNA molecules describedherein in the manufacture of a medicament for treating a disease.

Short Interfering Nucleic Acid (siNA) Molecules

As indicated above, the present disclosure provides siNA moleculescomprising modified nucleotides. Any of the siNA molecules describedherein may be double-stranded siNA (ds-siNA) molecules. The terms “siNAmolecules” and “ds-siNA molecules” may be used interchangeably. In someembodiments, the ds-siNA molecules comprise a sense strand and anantisense strand.

Further disclosed herein are siNA molecules comprising (a) at least onephosphorylation blocker, conjugated moiety, or 5′-stabilized end cap;and (b) a short interfering nucleic acid (siNA). In some embodiments,the phosphorylation blocker is a phosphorylation blocker disclosedherein. In some embodiments, the conjugated moiety is a galactosaminedisclosed herein. In some embodiments, the 5′-stabilized end cap is a5′-stabilized end cap disclosed herein. The siNA may comprise any of thefirst nucleotide, second nucleotide, sense strand, or antisense strandsequences disclosed herein. The siNA may comprise 5 to 100, 5 to 90, 10to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 30, 10to 25, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15to 30, or 15 to 25 nucleotides. The siNA may comprise at least 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. The siNAmay comprise less than or equal to 50, 45, 40, 39, 38, 37, 36, 35, 34,33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19nucleotides. The nucleotides may be modified nucleotides. The siNA maybe single stranded. The siNA may be double stranded. The siNA maycomprise (a) a sense strand comprising 15 to 30, 15 to 25, 15 to 24, 15to 23, 15 to 22, 15 to 21, 17 to 30, 17 to 25, 17 to 24, 17 to 23, 17 to22, 17 to 21, 18 to 30, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to21, 19 to 30, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 20 to25, 20 to 24, 20 to 23, 21 to 25, 21 to 24, or 21 to 23 nucleotides; and(b) an antisense strand comprising 15 to 30, 15 to 25, 15 to 24, 15 to23, 15 to 22, 15 to 21, 17 to 30, 17 to 25, 17 to 24, 17 to 23, 17 to22, 17 to 21, 18 to 30, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to21, 19 to 30, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 20 to25, 20 to 24, 20 to 23, 21 to 25, 21 to 24, or 21 to 23 nucleotides. ThesiNA may comprise (a) a sense strand comprising about 15, 16, 17, 18,19, 20, 21, 22, or 23 nucleotides; and (b) an antisense strandcomprising about 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides. ThesiNA may comprise (a) a sense strand comprising about 19 nucleotides;and (b) an antisense strand comprising about 21 nucleotides. The siNAmay comprise (a) a sense strand comprising about 21 nucleotides; and (b)an antisense strand comprising about 23 nucleotides.

In some embodiments, any of the siNA molecules disclosed herein furthercomprise one or more linkers independently selected from aphosphodiester (PO) linker, phosphorothioate (PS) linker,phosphorodithioate linker, and PS-mimic linker. In some embodiments, thePS-mimic linker is a sulfur linker. In some embodiments, the linkers areinternucleoside linkers. Alternatively, or additionally, the linkersconnect a nucleotide of the siNA molecule to at least onephosphorylation blocker, conjugated moiety, or 5′-stabilized end cap. Insome embodiments, the linkers connect a conjugated moiety to aphosphorylation blocker or 5′-stabilized end cap.

siNA Sense Strand

Any of the siNA molecules described herein may comprise a sense strand.The sense strand may comprise a first nucleotide sequence. The firstnucleotide sequence may be 15 to 30, 15 to 25, 15 to 23, 17 to 23, 19 to23, or 19 to 21 nucleotides in length. In some embodiments, the firstnucleotide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides in length. In some embodiments, the firstnucleotide sequence is at least 19 nucleotides in length. In someembodiments, the first nucleotide sequence is at least 21 nucleotides inlength.

In some embodiments, the sense strand is the same length as the firstnucleotide sequence. In some embodiments, the sense strand is longerthan the first nucleotide sequence. In some embodiments, the sensestrand may further comprise 1, 2, 3, 4, or 5 or more nucleotides thanthe first nucleotide sequence. In some embodiments, the sense strand mayfurther comprise a deoxyribonucleic acid (DNA). In some embodiments, theDNA is thymine (T). In some embodiments, the sense strand may furthercomprise a TT sequence. In some embodiments, the sense strand mayfurther comprise one or more modified nucleotides that are adjacent tothe first nucleotide sequence. In some embodiments, the one or moremodified nucleotides are independently selected from any of the modifiednucleotides disclosed herein (e.g., 2′-fluoro nucleotide, 2′-O-methylnucleotide, 2′-fluoro nucleotide mimic, 2′-O-methyl nucleotide mimic, ora nucleotide comprising a modified nucleobase).

In some embodiments, the first nucleotide sequence comprises 15, 16, 17,18, 19, 20, 21, 22, 23, or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. Insome embodiments, 70%, 75%, 80%, 85%, 90%, 95% or 100% of thenucleotides in the first nucleotide sequence are modified nucleotidesindependently selected from a 2′-O-methyl nucleotide and a 2′-fluoronucleotide. In some embodiments, 100% of the nucleotides in the firstnucleotide sequence are modified nucleotides independently selected froma 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. In someembodiments, the 2′-O-methyl nucleotide is a 2′-O-methyl nucleotidemimic. In some embodiments, the 2′-fluoro nucleotide is a 2′-fluoronucleotide mimic.

In some embodiments, between about 15 to 30, 15 to 25, 15 to 24, 15 to23, 15 to 22, 15 to 21, 17 to 30, 17 to 25, 17 to 24, 17 to 23, 17 to22, 17 to 21, 18 to 30, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to21, 19 to 30, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 20 to25, 20 to 24, 20 to 23, 21 to 25, 21 to 24, or 21 to 23 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 2 to 20 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 5 to 25 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 10 to 25 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 12 to 25 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least about 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or 22 modified nucleotides of the firstnucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, atleast about 12 modified nucleotides of the first nucleotide sequence are2′-O-methyl nucleotides. In some embodiments, at least about 13 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least about 14 modified nucleotidesof the first nucleotide sequence are 2′-O-methyl nucleotides. In someembodiments, at least about 15 modified nucleotides of the firstnucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, atleast about 16 modified nucleotides of the first nucleotide sequence are2′-O-methyl nucleotides. In some embodiments, at least about 17 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least about 18 modified nucleotidesof the first nucleotide sequence are 2′-O-methyl nucleotides. In someembodiments, at least about 19 modified nucleotides of the firstnucleotide sequence are 2′-O-methyl nucleotides. In some embodiments,less than or equal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of thefirst nucleotide sequence are 2′-O-methyl nucleotides. In someembodiments, less than or equal to 21 modified nucleotides of the firstnucleotide sequence are 2′-O-methyl nucleotides. In some embodiments,less than or equal to 20 modified nucleotides of the first nucleotidesequence are 2′-O-methyl nucleotides. In some embodiments, less than orequal to 19 modified nucleotides of the first nucleotide sequence are2′-O-methyl nucleotides. In some embodiments, less than or equal to 18modified nucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 17 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 16 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 15 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 14 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 13 modifiednucleotides of the first nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least one modified nucleotide ofthe first nucleotide sequence is a 2′-O-methyl pyrimidine. In someembodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of thefirst nucleotide sequence are 2′-O-methyl pyrimidines. In someembodiments, at least one modified nucleotide of the first nucleotidesequence is a 2′-O-methyl purine. In some embodiments, at least 5, 6, 7,8, 9, or 10 modified nucleotides of the first nucleotide sequence are2′-O-methyl purines. In some embodiments, the 2′-O-methyl nucleotide isa 2′-O-methyl nucleotide mimic.

In some embodiments, between 2 to 15 modified nucleotides of the firstnucleotide sequence are 2′-fluoro nucleotides. In some embodiments,between 2 to 10 modified nucleotides of the first nucleotide sequenceare 2′-fluoro nucleotides. In some embodiments, between 2 to 6 modifiednucleotides of the first nucleotide sequence are 2′-fluoro nucleotides.In some embodiments, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 modifiednucleotides of the first nucleotide sequence are 2′-fluoro nucleotides.In some embodiments, at least 1, 2, 3, 4, 5, or 6 modified nucleotidesof the first nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least 1 modified nucleotide of the first nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, at least 2modified nucleotides of the first nucleotide sequence are 2′-fluoronucleotides. In some embodiments, at least 3 modified nucleotides of thefirst nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least 4 modified nucleotides of the first nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, at least 5modified nucleotides of the first nucleotide sequence are 2′-fluoronucleotides. In some embodiments, at least 6 modified nucleotides of thefirst nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, 10, 9, 8, 7, 6, 5, 4, 3 or fewer modified nucleotides ofthe first nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, 10 or fewer modified nucleotides of the first nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, 7 or fewermodified nucleotides of the first nucleotide sequence are 2′-fluoronucleotides. In some embodiments, 6 or fewer modified nucleotides of thefirst nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, 5 or fewer modified nucleotides of the first nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, 4 or fewermodified nucleotides of the first nucleotide sequence are 2′-fluoronucleotides. In some embodiments, 3 or fewer modified nucleotides of thefirst nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, 2 or fewer modified nucleotides of the first nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, at least onemodified nucleotide of the first nucleotide sequence is a 2′-fluoropyrimidine. In some embodiments, 1, 2, 3, 4, 5, or 6 modifiednucleotides of the first nucleotide sequence are 2′-fluoro pyrimidines.In some embodiments, at least one modified nucleotide of the firstnucleotide sequence is a 2′-fluoro purine. In some embodiments, 1, 2, 3,4, 5, or 6 modified nucleotides of the first nucleotide sequence are2′-fluoro purines. In some embodiments, the 2′-fluoro nucleotide is a2′-fluoro nucleotide mimic.

In some embodiments, the nucleotide at position 3, 5, 7, 8, 9, 10, 11,12, 14, 17, and/or 19 from the 5′ end of the first nucleotide sequenceis a 2′-fluoro nucleotide. In some embodiments, at least two nucleotidesat positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′end of the first nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least three nucleotides at positions 3, 5, 7, 8, 9, 10,11, 12, 14, 17, and/or 19 from the 5′ end of the first nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, at least fournucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19from the 5′ end of the first nucleotide sequence are 2′-fluoronucleotides. In some embodiments, at least five nucleotides at positions3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end of thefirst nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14,17, and/or 19 from the 5′ end of the first nucleotide sequence are2′-fluoro nucleotides. In some embodiments, the nucleotide at position 3from the 5′ end of the first nucleotide sequence is a 2′-fluoronucleotide. In some embodiments, the nucleotide at position 7 from the5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. Insome embodiments, the nucleotide at position 8 from the 5′ end of thefirst nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at position 9 from the 5′ end of the firstnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, thenucleotide at position 12 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 17 from the 5′ end of the first nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, the 2′-fluoro nucleotide is a2′-fluoro nucleotide mimic.

In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 nucleotides atposition 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end ofthe first nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at positions 3, 5, 7, 8, 9, 10, 11, 12, 14,17, and/or 19 from the 5′ end of the first nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, at least two nucleotides atpositions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ endof the first nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least three nucleotides at positions 3, 5, 7, 8, 9, 10,11, 12, 14, 17, and/or 19 from the 5′ end of the first nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, the nucleotidesat positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′end of the first nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, the nucleotide at position 3 from the 5′ end of the firstnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, thenucleotide at position 5 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 7 from the 5′ end of the first nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, the nucleotide at position 8from the 5′ end of the first nucleotide sequence is a 2′-fluoronucleotide. In some embodiments, the nucleotide at position 9 from the5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. Insome embodiments, the nucleotide at position 10 from the 5′ end of thefirst nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at position 11 from the 5′ end of the firstnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, thenucleotide at position 12 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 14 from the 5′ end of the first nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, the nucleotide at position 17from the 5′ end of the first nucleotide sequence is a 2′-fluoronucleotide. In some embodiments, the nucleotide at position 19 from the5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. Insome embodiments, the nucleotide at position 3, 7, 8, 9, 12, and/or 17from the 5′ end of the first nucleotide sequence is a 2′-fluoronucleotide. In some embodiments, the nucleotide at position 3, 7, 8,and/or 17 from the 5′ end of the first nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, the nucleotide at position 3,7, 8, 9, 12, and/or 17 from the 5′ end of the first nucleotide sequenceis a 2′-fluoro nucleotide. In some embodiments, the nucleotide atposition 5, 7, 8, and/or 9 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 5, 9, 10, 11, 12, and/or 19 from the 5′ end of the firstnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, the2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic.

In some embodiments, the 2′-fluoro nucleotide or 2′-O-methyl nucleotideis a 2′-fluoro or 2′-O-methyl nucleotide mimic. In some embodiments, the2′-fluoro or 2′-O—methyl nucleotide mimic is a nucleotide mimic ofFormula (V):

wherein R¹ is independently a nucleobase, aryl, heteroaryl, or H, Q¹ andQ² are independently S or O, R⁵ is independently —OCD₃, —F, or —OCH₃,and R⁶ and R⁷ are independently H, D, or CD3. In some embodiments, thenucleobase is selected from cytosine, guanine, adenine, uracil, aryl,heteroaryl, and an analogue or derivative thereof.

In some embodiments, the 2′-fluoro or 2′-O-methyl nucleotide mimic is anucleotide mimic of Formula (16) -Formula (20):

wherein R¹ is independently a nucleobase and R² is F or —OCH₃. In someembodiments, the nucleobase is selected from cytosine, guanine, adenine,uracil, aryl, heteroaryl, and an analogue or derivative thereof.

In some embodiments, the first nucleotide sequence comprises, consistsof, or consists essentially of ribonucleic acids (RNAs). In someembodiments, the first nucleotide sequence comprises, consists of, orconsists essentially of modified RNAs. In some embodiments, the modifiedRNAs are selected from a 2′-O-methyl RNA and 2′-fluoro RNA. In someembodiments, 15, 16, 17, 18, 19, 20, 21, 22, or 23 modified nucleotidesof the first nucleotide sequence are independently selected from2′-O-methyl RNA and 2′-fluoro RNA.

In some embodiments, the sense strand may further comprise one or moreinternucleoside linkages independently selected from a phosphodiester(PO) internucleoside linkage, phosphorothioate (PS) internucleosidelinkage, phosphorodithioate internucleoside linkage, and PS-mimicinternucleoside linkage. In some embodiments, the PS-mimicinternucleoside linkage is a sulfo internucleoside linkage.

In some embodiments, the sense strand may further comprise at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or morephosphorothioate internucleoside linkages. In some embodiments, thesense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, or 3 or fewer phosphorothioate internucleoside linkages. Insome embodiments, the sense strand comprises 2 to 10, 2 to 8, 2 to 6, 1to 5, 1 to 4, 1 to 3, or 1 to 2 phosphorothioate internucleosidelinkages. In some embodiments, the sense strand comprises 1 to 2phosphorothioate internucleoside linkages. In some embodiments, thesense strand comprises 2 to 4 phosphorothioate internucleoside linkages.In some embodiments, at least one phosphorothioate internucleosidelinkage is between the nucleotides at positions 1 and 2 from the 5′ endof the first nucleotide sequence. In some embodiments, at least onephosphorothioate internucleoside linkage is between the nucleotides atpositions 2 and 3 from the 5′ end of the first nucleotide sequence. Insome embodiments, the sense strand comprises two phosphorothioateinternucleoside linkages between the nucleotides at positions 1 to 3from the 5′ end of the first nucleotide sequence.

In some embodiments, any of the sense strands disclosed herein furthercomprise a monomer selected from Examples 21-32, 36, 37, 40-42, and44-46 monomers. In some embodiments, any of the sense strands disclosedherein further comprise a 5′ end cap monomer. In some embodiments, the5′ end cap monomer is selected from Examples 5-11, 33-35, 38, 39, 43,and 49-53 5′ end cap monomers.

In some embodiments, any of the first nucleotide sequences disclosedherein further comprise a monomer selected from Examples 21-32, 36, 37,40-42, and 44-46 monomers. In some embodiments, any of the firstnucleotide sequences disclosed herein further comprise a 5′ end capmonomer. In some embodiments, the 5′ end cap monomer is selected fromExamples 5-11, 33-35, 38, 39, 43, and 49-53 5′ end cap monomers.

siNA Antisense Strand

Any of the siNA molecules described herein may comprise an antisensestrand. The antisense strand may comprise a second nucleotide sequence.The second nucleotide sequence may be 15 to 30, 15 to 25, 15 to 23, 17to 23, 19 to 23, or 19 to 21 nucleotides in length. In some embodiments,the second nucleotide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In someembodiments, the second nucleotide sequence is at least 19 nucleotidesin length. In some embodiments, the second nucleotide sequence is atleast 21 nucleotides in length.

In some embodiments, the antisense strand is the same length as thesecond nucleotide sequence. In some embodiments, the antisense strand islonger than the second nucleotide sequence. In some embodiments, theantisense strand may further comprise 1, 2, 3, 4, or 5 or morenucleotides than the second nucleotide sequence. In some embodiments,the antisense strand is the same length as the sense strand. In someembodiments, the antisense strand is longer than the sense strand. Insome embodiments, the antisense strand may further comprise 1, 2, 3, 4,or 5 or more nucleotides than the sense strand. In some embodiments, theantisense strand may further comprise a deoxyribonucleic acid (DNA). Insome embodiments, the DNA is thymine (T). In some embodiments, theantisense strand may further comprise a TT sequence. In someembodiments, the antisense strand may further comprise one or moremodified nucleotides that are adjacent to the second nucleotidesequence. In some embodiments, the one or more modified nucleotides areindependently selected from any of the modified nucleotides disclosedherein (e.g., 2′-fluoro nucleotide, 2′-O-methyl nucleotide, 2′-fluoronucleotide mimic, 2′-O-methyl nucleotide mimic, or a nucleotidecomprising a modified nucleobase).

In some embodiments, the second nucleotide sequence comprises 15, 16,17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independentlyselected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. Insome embodiments, 70%, 75%, 80%, 85%, 90%, 95% or 100% of thenucleotides in the second nucleotide sequence are modified nucleotidesindependently selected from a 2′-O-methyl nucleotide and a 2′-fluoronucleotide. In some embodiments, 100% of the nucleotides in the secondnucleotide sequence are modified nucleotides independently selected froma 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.

In some embodiments, between about 15 to 30, 15 to 25, 15 to 24, 15 to23, 15 to 22, 15 to 21, 17 to 30, 17 to 25, 17 to 24, 17 to 23, 17 to22, 17 to 21, 18 to 30, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to21, 19 to 30, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 20 to25, 20 to 24, 20 to 23, 21 to 25, 21 to 24, or 21 to 23 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 2 to 20 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 5 to 25 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 10 to 25 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, between about 12 to 25 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, or 22 modified nucleotides of the second nucleotidesequence are 2′-O-methyl nucleotides. In some embodiments, at leastabout 12 modified nucleotides of the second nucleotide sequence are2′-O-methyl nucleotides. In some embodiments, at least about 13 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least about 14 modified nucleotidesof the second nucleotide sequence are 2′-O-methyl nucleotides. In someembodiments, at least about 15 modified nucleotides of the secondnucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, atleast about 16 modified nucleotides of the second nucleotide sequenceare 2′-O-methyl nucleotides. In some embodiments, at least about 17modified nucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least about 18 modified nucleotidesof the second nucleotide sequence are 2′-O-methyl nucleotides. In someembodiments, at least about 19 modified nucleotides of the secondnucleotide sequence are 2′-O-methyl nucleotides. In some embodiments,less than or equal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of thesecond nucleotide sequence are 2′-O-methyl nucleotides. In someembodiments, less than or equal to 21 modified nucleotides of the secondnucleotide sequence are 2′-O-methyl nucleotides. In some embodiments,less than or equal to 20 modified nucleotides of the second nucleotidesequence are 2′-O-methyl nucleotides. In some embodiments, less than orequal to 19 modified nucleotides of the second nucleotide sequence are2′-O-methyl nucleotides. In some embodiments, less than or equal to 18modified nucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 17 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 16 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 15 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 14 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, less than or equal to 13 modifiednucleotides of the second nucleotide sequence are 2′-O-methylnucleotides. In some embodiments, at least one modified nucleotide ofthe second nucleotide sequence is a 2′-O-methyl pyrimidine. In someembodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of thesecond nucleotide sequence are 2′-O-methyl pyrimidines. In someembodiments, at least one modified nucleotide of the second nucleotidesequence is a 2′-O-methyl purine. In some embodiments, at least 5, 6, 7,8, 9, or 10 modified nucleotides of the second nucleotide sequence are2′-O-methyl purines. In some embodiments, the 2′-O-methyl nucleotide isa 2′-O-methyl nucleotide mimic.

In some embodiments, between 2 to 15 modified nucleotides of the secondnucleotide sequence are 2′-fluoro nucleotides. In some embodiments,between 2 to 10 modified nucleotides of the second nucleotide sequenceare 2′-fluoro nucleotides. In some embodiments, between 2 to 6 modifiednucleotides of the second nucleotide sequence are 2′-fluoro nucleotides.In some embodiments, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 modifiednucleotides of the second nucleotide sequence are 2′-fluoro nucleotides.In some embodiments, at least 1, 2, 3, 4, 5, or 6 modified nucleotidesof the second nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least 1 modified nucleotide of the second nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, at least 2modified nucleotides of the second nucleotide sequence are 2′-fluoronucleotides. In some embodiments, at least 3 modified nucleotides of thesecond nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least 4 modified nucleotides of the second nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, at least 5modified nucleotides of the second nucleotide sequence are 2′-fluoronucleotides. In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3 or fewermodified nucleotides of the second nucleotide sequence are 2′-fluoronucleotides. In some embodiments, 10 or fewer modified nucleotides ofthe second nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, 7 or fewer modified nucleotides of the second nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, 6 or fewermodified nucleotides of the second nucleotide sequence are 2′-fluoronucleotides. In some embodiments, 5 or fewer modified nucleotides of thesecond nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, 4 or fewer modified nucleotides of the second nucleotidesequence are 2′-fluoro nucleotides. In some embodiments, 3 or fewermodified nucleotides of the second nucleotide sequence are 2′-fluoronucleotides. In some embodiments, 2 or fewer modified nucleotides of thesecond nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least one modified nucleotide of the second nucleotidesequence is a 2′-fluoro pyrimidine. In some embodiments, 1, 2, 3, 4, 5,or 6 modified nucleotides of the second nucleotide sequence are2′-fluoro pyrimidines. In some embodiments, at least one modifiednucleotide of the second nucleotide sequence is a 2′-fluoro purine. Insome embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides of the secondnucleotide sequence are 2′-fluoro purines. In some embodiments, the2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic.

In some embodiments, the 2′-fluoro nucleotide or 2′-O-methyl nucleotideis a 2′-fluoro or 2′-O-methyl nucleotide mimic. In some embodiments, the2′-fluoro or 2′-O—methyl nucleotide mimic is a nucleotide mimic ofFormula (V):

wherein R¹ is independently a nucleobase, aryl, heteroaryl, or H, Q¹ andQ² are independently S or O, R⁵ is independently —OCD₃, —F, or —OCH₃,and R⁶ and R⁷ are independently H, D, or CD3. In some embodiments, thenucleobase is selected from cytosine, guanine, adenine, uracil, aryl,heteroaryl, and an analogue or derivative thereof.

In some embodiments, the 2′-fluoro or 2′-O-methyl nucleotide mimic is anucleotide mimic of Formula (16) -Formula (20):

wherein R¹ is a nucleobase and R² is independently F or —OCH₃. In someembodiments, the nucleobase is selected from cytosine, guanine, adenine,uracil, aryl, heteroaryl, and an analogue or derivative thereof.

In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotidesat position 2, 5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of thesecond nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at position 2, 5, 6, 8, 10, 14, 16, 17,and/or 18 from the 5′ end of the second nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, at least two nucleotides atpositions 2, 5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of thesecond nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least three nucleotides at positions 2, 5, 6, 8, 10, 14,16, 17, and/or 18 from the 5′ end of the second nucleotide sequence are2′-fluoro nucleotides. In some embodiments, at least four nucleotides atpositions 2, 5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of thesecond nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, at least five nucleotides at positions 2, 5, 6, 8, 10, 14,16, 17, and/or 18 from the 5′ end of the second nucleotide sequence are2′-fluoro nucleotides. In some embodiments, the nucleotides at positions2 and/or 14 from the 5′ end of the second nucleotide sequence are2′-fluoro nucleotides. In some embodiments, the nucleotides at positions2, 6, and/or 16 from the 5′ end of the second nucleotide sequence are2′-fluoro nucleotides. In some embodiments, the nucleotides at positions2, 6, 14, and/or 16 from the 5′ end of the second nucleotide sequenceare 2′-fluoro nucleotides. In some embodiments, the nucleotides atpositions 2, 6, 10, 14, and/or 18 from the 5′ end of the secondnucleotide sequence are 2′-fluoro nucleotides. In some embodiments, thenucleotides at positions 2, 5, 8, 14, and/or 17 from the 5′ end of thesecond nucleotide sequence are 2′-fluoro nucleotides. In someembodiments, the nucleotide at position 2 from the 5′ end of the secondnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, thenucleotide at position 5 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 6 from the 5′ end of the second nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, the nucleotide at position 8from the 5′ end of the second nucleotide sequence is a 2′-fluoronucleotide. In some embodiments, the nucleotide at position 10 from the5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide. Insome embodiments, the nucleotide at position 14 from the 5′ end of thesecond nucleotide sequence is a 2′-fluoro nucleotide. In someembodiments, the nucleotide at position 16 from the 5′ end of the secondnucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, thenucleotide at position 17 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotideat position 18 from the 5′ end of the second nucleotide sequence is a2′-fluoro nucleotide. In some embodiments, the 2′-fluoro nucleotide is a2′-fluoro nucleotide mimic.

In some embodiments, the nucleotides in the second nucleotide sequenceare arranged in an alternating 1:3 modification pattern, wherein 1nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methylnucleotides, and wherein the alternating 1:3 modification pattern occursat least 2 times. In some embodiments, the alternating 1:3 modificationpattern occurs 2-5 times. In some embodiments, at least two of thealternating 1:3 modification pattern occur consecutively. In someembodiments, at least two of the alternating 1:3 modification patternoccurs nonconsecutively. In some embodiments, at least 1, 2, 3, 4, or 5alternating 1:3 modification pattern begins at nucleotide position 2, 6,10, 14, and/or 18 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:3 modification pattern begins atnucleotide position 2 from the 5′ end of the antisense strand. In someembodiments, wherein at least one alternating 1:3 modification patternbegins at nucleotide position 6 from the 5′ end of the antisense strand.In some embodiments, at least one alternating 1:3 modification patternbegins at nucleotide position 10 from the 5′ end of the antisensestrand. In some embodiments, at least one alternating 1:3 modificationpattern begins at nucleotide position 14 from the 5′ end of theantisense strand. In some embodiments, at least one alternating 1:3modification pattern begins at nucleotide position 18 from the 5′ end ofthe antisense strand. In some embodiments, the 2′-fluoro nucleotide is a2′-fluoro nucleotide mimic.

In some embodiments, the nucleotides in the second nucleotide sequenceare arranged in an alternating 1:2 modification pattern, wherein 1nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methylnucleotides, and wherein the alternating 1:2 modification pattern occursat least 2 times. In some embodiments, the alternating 1:2 modificationpattern occurs 2-5 times. In some embodiments, at least two of thealternating 1:2 modification pattern occurs consecutively. In someembodiments, at least two of the alternating 1:2 modification patternoccurs nonconsecutively. In some embodiments, at least 1, 2, 3, 4, or 5alternating 1:2 modification pattern begins at nucleotide position 2, 5,8, 14, and/or 17 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 2 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 5 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 8 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 14 from the 5′ end of the antisense strand. In someembodiments, at least one alternating 1:2 modification pattern begins atnucleotide position 17 from the 5′ end of the antisense strand. In someembodiments, the 2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic.

In some embodiments, the second nucleotide sequence comprises, consistsof, or consists essentially of ribonucleic acids (RNAs). In someembodiments, the second nucleotide sequence comprises, consists of, orconsists essentially of modified RNAs. In some embodiments, the modifiedRNAs are selected from a 2′-O-methyl RNA and 2′-fluoro RNA. In someembodiments, 15, 16, 17, 18, 19, 20, 21, 22, or 23 modified nucleotidesof the second nucleotide sequence are independently selected from2′-O-methyl RNA and 2′-fluoro RNA. In some embodiments, the 2′-fluoronucleotide is a 2′-fluoro nucleotide mimic.

In some embodiments, the sense strand may further comprise one or moreinternucleoside linkages independently selected from a phosphodiester(PO) internucleoside linkage, phosphorothioate (PS) internucleosidelinkage, phosphorodithioate internucleoside linkage, and PS-mimicinternucleoside linkage. In some embodiments, the PS-mimicinternucleoside linkage is a sulfo internucleoside linkage.

In some embodiments, the antisense strand may further comprise at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or morephosphorothioate internucleoside linkages. In some embodiments, theantisense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, or 3 or fewer phosphorothioate internucleosidelinkages. In some embodiments, the antisense strand comprises 2 to 10, 2to 8, 2 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 phosphorothioateinternucleoside linkages. In some embodiments, the antisense strandcomprises 2 to 10, 2 to 8, 2 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2phosphorothioate internucleoside linkages. In some embodiments, theantisense strand comprises 2 to 8 phosphorothioate internucleosidelinkages. In some embodiments, the antisense strand comprises 3 to 8phosphorothioate internucleoside linkages. In some embodiments, theantisense strand comprises 4 to 8 phosphorothioate internucleosidelinkages. In some embodiments, at least one phosphorothioateinternucleoside linkage is between the nucleotides at positions 1 and 2from the 5′ end of the second nucleotide sequence. In some embodiments,at least one phosphorothioate internucleoside linkage is between thenucleotides at positions 2 and 3 from the 5′ end of the secondnucleotide sequence. In some embodiments, at least one phosphorothioateinternucleoside linkage is between the nucleotides at positions 1 and 2from the 3′ end of the second nucleotide sequence. In some embodiments,at least one phosphorothioate internucleoside linkage is between thenucleotides at positions 2 and 3 from the 3′ end of the secondnucleotide sequence. In some embodiments, the antisense strand comprisestwo phosphorothioate internucleoside linkages between the nucleotides atpositions 1 to 3 from the 5′ end of the first nucleotide sequence. Insome embodiments, the antisense strand comprises two phosphorothioateinternucleoside linkages between the nucleotides at positions 1 to 3from the 3′ end of the first nucleotide sequence. In some embodiments,the antisense strand comprises (a) two phosphorothioate internucleosidelinkages between the nucleotides at positions 1 to 3 from the 5′ end ofthe first nucleotide sequence; and (b) two phosphorothioateinternucleoside linkages between the nucleotides at positions 1 to 3from the 3′ end of the first nucleotide sequence.

In some embodiments, at least one end of the ds-siNA is a blunt end. Insome embodiments, at least one end of the ds-siNA comprises an overhang,wherein the overhang comprises at least one nucleotide. In someembodiments, both ends of the ds-siNA comprise an overhang, wherein theoverhang comprises at least one nucleotide. In some embodiments, theoverhang comprises 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3nucleotides, or 1 to 2 nucleotides. In some embodiments, the overhangconsists of 1 to 2 nucleotides.

In some embodiments, the first nucleotide sequence comprises anucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.In some embodiments, the second nucleotide sequence comprises anucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and261-306. In some embodiments, the sense strand comprises a nucleotidesequence of any one of SEQ ID NOs: 307-362 and 415-444. In someembodiments, the antisense strand comprises a nucleotide sequence of anyone of SEQ ID NOs: 363-409, 445-533, and 536-539.

In some embodiments, any of the antisense strands disclosed hereinfurther comprise a monomer selected from Examples 21-32, 36, 37, 40-42,and 44-46 monomers. In some embodiments, any of the antisense strandsdisclosed herein further comprise a 5′ end cap monomer. In someembodiments, the 5′ end cap monomer is selected from Examples 5-11,33-35, 38, 39, 43, and 49-53 5′ end cap monomers.

In some embodiments, any of the secondnucleotide sequences disclosedherein further comprise a monomer selected from Examples 21-32, 36, 37,40-42, and 44-46 monomers. In some embodiments, any of the secondnucleotide sequences disclosed herein further comprise a 5′ end capmonomer. In some embodiments, the 5′ end cap monomer is selected fromExamples 5-11, 33-35, 38, 39, 43, and 49-53 5′ end cap monomers.

Modified Nucleotides

Further disclosed herein are siNA molecules comprising one or moremodified nucleotides. In some embodiments, any of the siNAs disclosedherein comprise one or more modified nucleotides. In some embodiments,any of the sense strands disclosed herein comprise one or more modifiednucleotides. In some embodiments, any of the first nucleotide sequencesdisclosed herein comprise one or more modified nucleotides. In someembodiments, any of the antisense strands disclosed herein comprise oneor more modified nucleotides. In some embodiments, any of the secondnucleotide sequences disclosed herein comprise one or more modifiednucleotides. In some embodiments, the one or more modified nucleotidesis adjacent to the first nucleotide sequence. In some embodiments, atleast one modified nucleotide is adjacent to the 5′ end of the firstnucleotide sequence. In some embodiments, at least one modifiednucleotide is adjacent to the 3′ end of the first nucleotide sequence.In some embodiments, at least one modified nucleotide is adjacent to the5′ end of the first nucleotide sequence and at least one modifiednucleotide is adjacent to the 3′ end of the first nucleotide sequence.In some embodiments, the one or more modified nucleotides is adjacent tothe second nucleotide sequence. In some embodiments, at least onemodified nucleotide is adjacent to the 5′ end of the second nucleotidesequence. In some embodiments, at least one modified nucleotide isadjacent to the 3′ end of the second nucleotide sequence. In someembodiments, at least one modified nucleotide is adjacent to the 5′ endof the second nucleotide sequence and at least one modified nucleotideis adjacent to the 3′ end of the second nucleotide sequence. In someembodiments, a 2′-O-methyl nucleotide in any of sense strands or firstnucleotide sequences disclosed herein is replaced with a modifiednucleotide. In some embodiments, a 2′-O-methyl nucleotide in any ofantisense strands or second nucleotide sequences disclosed herein isreplaced with a modified nucleotide.

In some embodiments, any of the siNA molecules, siNAs, sense strands,first nucleotide sequences, antisense strands, and second nucleotidesequences disclosed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 or more modified nucleotides. In some embodiments, 1%, 2%, 3%, 4%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%,80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% of the nucleotides in the siNA molecule, siNA, sense strand,first nucleotide sequence, antisense strand, or second nucleotidesequence are modified nucleotides.

In some embodiments, a modified nucleotide is selected from the groupconsisting of 2′-fluoro nucleotide, 2′-O-methyl nucleotide, 2′-fluoronucleotide mimic, 2′-O-methyl nucleotide mimic, a locked nucleic acid,and a nucleotide comprising a modified nucleobase.

In some embodiments, any of the siRNAs disclosed herein comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2′-fluoro or 2′-O-methylnucleotide mimics. In some embodiments, any of the sense strandsdisclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore 2′-fluoro or 2′-O-methyl nucleotide mimics. In some embodiments,any of the first nucleotide sequences disclosed herein comprise at least1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2′-fluoro or 2′-O-methylnucleotide mimics. In some embodiments, any of the antisense stranddisclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore 2′-fluoro or 2′-O-methyl nucleotide mimics. In some embodiments,any of the second nucleotide sequences disclosed herein comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2′-fluoro or 2′-O-methylnucleotide mimics. In some embodiments, the 2′-fluoro or 2′-O-methylnucleotide mimic is a nucleotide mimic of Formula (16) -Formula (20):

wherein R¹ is a nucleobase and R² is independently F or —OCH₃. In someembodiments, the nucleobase is selected from cytosine, guanine, adenine,uracil, aryl, heteroaryl, and an analogue or derivative thereof. In someembodiments, the siNA molecules disclosed herein comprise at least one2′-fluoro nucleotide, at least one 2′-O-methyl nucleotide, and at leastone 2′-fluoro or 2′-O-methyl nucleotide mimic. In some embodiments, theat least one 2′-fluoro or 2′-O-methyl nucleotide mimic is adjacent tothe first nucleotide sequence. In some embodiments, the at least one2′-fluoro or 2′-O-methyl nucleotide mimic is adjacent to the 5′ end offirst nucleotide sequence. In some embodiments, the at least one2′-fluoro or 2′-O-methyl nucleotide mimic is adjacent to the 3′ end offirst nucleotide sequence. In some embodiments, the at least one2′-fluoro or 2′-O-methyl nucleotide mimic is adjacent to the secondnucleotide sequence. In some embodiments, the at least one 2′-fluoro or2′-O-methyl nucleotide mimic is adjacent to the 5′ end of secondnucleotide sequence. In some embodiments, the at least one 2′-fluoro or2′-O-methyl nucleotide mimic is adjacent to the 3′ end of secondnucleotide sequence. In some embodiments, the first nucleotide sequencedoes not comprise a 2′-fluoro nucleotide mimic. In some embodiments, thefirst nucleotide sequence does not comprise a 2′-O-methyl nucleotidemimic. In some embodiments, the second nucleotide sequence does notcomprise a 2′-fluoro nucleotide mimic. In some embodiments, the secondnucleotide sequence does not comprise a 2′-O-methyl nucleotide mimic.

In some embodiments, any of the siRNAs disclosed herein comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more locked nucleic acids. Insome embodiments, any of the sense strands disclosed herein comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more locked nucleic acids. Insome embodiments, any of the first nucleotide sequences disclosed hereincomprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more lockednucleic acids. In some embodiments, any of the antisense stranddisclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore locked nucleic acids. In some embodiments, any of the secondnucleotide sequences disclosed herein comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more locked nucleic acids. In some embodiments, thelocked nucleic acid is selected from

where R is H or alkyl (or AmNA(N-Me)) when R is alkyl);

wherein B is a nucleobase. In some embodiments, any of the siRNAs, sensestrands, first nucleotide sequences, antisense strands, or secondnucleotide sequences disclosed herein comprise at least modifiednucleotide that is

In some embodiments, any of the siRNAs, sense strands, first nucleotidesequences, antisense strands, or second nucleotide sequences disclosedherein comprise at least modified nucleotide that is

In some embodiments, any of the siRNAs, sense strands, first nucleotidesequences, antisense strands, or second nucleotide sequences disclosedherein comprise at least modified nucleotide that is

where R is H or alkyl (or AmNA(N-Me)) when R is alkyl). In someembodiments, any of the siRNAs, sense strands, first nucleotidesequences, antisense strands, or second nucleotide sequences disclosedherein comprise at least modified nucleotide that is

In some embodiments, any of the siRNAs, sense strands, first nucleotidesequences, antisense strands, or second nucleotide sequences disclosedherein comprise at least modified nucleotide that is

wherein B is a nucleobase.

Phosphorylation Blocker

Further disclosed herein are siNA molecules comprising a phosphorylationblocker. In some embodiments, a 2′-O-methyl nucleotide in any of sensestrands or first nucleotide sequences disclosed herein is replaced witha nucleotide containing a phosphorylation blocker. In some embodiments,a 2′-O-methyl nucleotide in any of antisense strands or secondnucleotide sequences disclosed herein is replaced with a nucleotidecontaining a phosphorylation blocker. In some embodiments, a 2′-O-methylnucleotide in any of sense strands or first nucleotide sequencesdisclosed herein is further modified to contain a phosphorylationblocker. In some embodiments, a 2′-O-methyl nucleotide in any ofantisense strands or second nucleotide sequences disclosed herein isfurther modified to contain a phosphorylation blocker.

In some embodiments, any of the siNA molecules disclosed herein comprisea phosphorylation blocker of Formula (IV):

wherein R¹ is a nucleobase, R⁴ is —O—R³⁰ or —NR³¹R³², R³⁰ is C₁-C₈substituted or unsubstituted alkyl; and R³¹ and R³² together with thenitrogen to which they are attached form a substituted or unsubstitutedheterocyclic ring.

In some embodiments, any of the siNA molecules disclosed herein comprisea phosphorylation blocker of Formula (IV):

wherein R¹ is a nucleobase, and R⁴ is —OCH₃ or —N(CH₂CH₂)₂O.

In some embodiments, a siNA molecule comprises (a) a phosphorylationblocker of Formula (IV):

wherein R¹ is a nucleobase, R⁴ is —O—R³⁰ or —NR³¹R³², R³⁰ is C₁-C₈substituted or unsubstituted alkyl; and R³¹ and R³² together with thenitrogen to which they are attached form a substituted or unsubstitutedheterocyclic ring; and (b) a short interfering nucleic acid (siNA),wherein the phosphorylation blocker is conjugated to the siNA.

In some embodiments, a siNA molecule comprises (a) a phosphorylationblocker of Formula (IV):

wherein R¹ is a nucleobase, and R⁴ is —OCH₃ or —N(CH₂CH₂)₂O; and (b) ashort interfering nucleic acid (siNA), wherein the phosphorylationblocker is conjugated to the siNA.

In some embodiments, the phosphorylation blocker is attached to the 3′end of the sense strand or first nucleotide sequence. In someembodiments, the phosphorylation blocker is attached to the 3′ end ofthe sense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 ormore linkers. In some embodiments, the phosphorylation blocker isattached to the 5′ end of the sense strand or first nucleotide sequence.In some embodiments, the phosphorylation blocker is attached to the 5′end of the sense strand or first nucleotide sequence via 1, 2, 3, 4, or5 or more linkers. In some embodiments, the phosphorylation blocker isattached to the 3′ end of the antisense strand or second nucleotidesequence. In some embodiments, the phosphorylation blocker is attachedto the 3′ end of the antisense strand or second nucleotide sequence via1, 2, 3, 4, or 5 or more linkers. In some embodiments, thephosphorylation blocker is attached to the 5′ end of the antisensestrand or second nucleotide sequence. In some embodiments, thephosphorylation blocker is attached to the 5′ end of the antisensestrand or second nucleotide sequence via 1, 2, 3, 4, or 5 or morelinkers. In some embodiments, the one or more linkers are independentlyselected from the group consisting of a phosphodiester linker,phosphorothioate linker, and phosphorodithioate linker.

Conjugated Moiety

Further disclosed herein are siNA molecules comprising a conjugatedmoiety. In some embodiments, the conjugated moiety is selected fromgalactosamine, peptides, proteins, sterols, lipids, phospholipids,biotin, phenoxazines, active drug substance, cholesterols,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. In some embodiments, the conjugated moiety isattached to the 3′ end of the sense strand or first nucleotide sequence.In some embodiments, the conjugated moiety is attached to the 3′ end ofthe sense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 ormore linkers. In some embodiments, the conjugated moiety is attached tothe 5′ end of the sense strand or first nucleotide sequence. In someembodiments, the conjugated moiety is attached to the 5′ end of thesense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 or morelinkers. In some embodiments, the conjugated moiety is attached to the3′ end of the antisense strand or second nucleotide sequence. In someembodiments, the conjugated moiety is attached to the 3′ end of theantisense strand or second nucleotide sequence via 1, 2, 3, 4, or 5 ormore linkers. In some embodiments, the conjugated moiety is attached tothe 5′ end of the antisense strand or second nucleotide sequence. Insome embodiments, the conjugated moiety is attached to the 5′ end of theantisense strand or second nucleotide sequence via 1, 2, 3, 4, or 5 ormore linkers. In some embodiments, the one or more linkers areindependently selected from the group consisting of a phosphodiesterlinker, phosphorothioate linker, and phosphorodithioate linker.

In some embodiments, the conjugated moiety is galactosamine. In someembodiments, any of the siNAs disclosed herein are attached to aconjugated moiety that is galactosamine In some embodiments, thegalactosamine is N-acetylgalactosamine (GalNAc). In some embodiments,any of the siNA molecules disclosed herein comprise GalNAc. In someembodiments, the GalNAc is of Formula (VI):

wherein m is 1, 2, 3, 4, or 5; each n is independently 1 or 2; p is 0 or1; each R is independently H or a first protecting group; each Y isindependently selected from —O—P(═O)(SH)—, —O—P(═O)(O)—, —O—P(═O)(OH)—,—O—P(S)S—, and —O—; Z is H or a second protecting group; either L is alinker or L and Y in combination are a linker; and A is H, OH, a thirdprotecting group, an activated group, or an oligonucleotide. In someembodiments, the first protecting group is acetyl. In some embodiments,the second protecting group is trimethoxytrityl (TMT). In someembodiments, the activated group is a phosphoramidite group. In someembodiments, the phosphoramidite group is a cyanoethoxyN,N-diisopropylphosphoramidite group. In some embodiments, the linker isa C6—NH₂ group. In some embodiments, A is a short interfering nucleicacid (siNA) or siNA molecule. In some embodiments, m is 3. In someembodiments, R is H, Z is H, and n is 1. In some embodiments, R is H, Zis H, and n is 2.

In some embodiments, the GalNAc is of Formula (VII):

wherein each n is independently 1 or 2.

In some embodiments, the galactosamine is attached to the 3′ end of thesense strand or first nucleotide sequence. In some embodiments, thegalactosamine is attached to the 3′ end of the sense strand or firstnucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In someembodiments, the galactosamine is attached to the 5′ end of the sensestrand or first nucleotide sequence. In some embodiments, thegalactosamine is attached to the 5′ end of the sense strand or firstnucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In someembodiments, the galactosamine is attached to the 3′ end of theantisense strand or second nucleotide sequence. In some embodiments, thegalactosamine is attached to the 3′ end of the antisense strand orsecond nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In someembodiments, the galactosamine is attached to the 5′ end of theantisense strand or second nucleotide sequence. In some embodiments, thegalactosamine is attached to the 5′ end of the antisense strand orsecond nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In someembodiments, the one or more linkers are independently selected from thegroup consisting of a phosphodiester (p or po) linker, phosphorothioate(ps) linker, phosphoramidite (HEG) linker, triethylene glycol (TEG)linker, and/or phosphorodithioate linker. In some embodiments, the oneor more linkers are independently selected from the group consisting ofp-(PS)2, (PS)2-p-TEG-p, (PS)2-p-HEG-p, and (PS)2-p-(HEG-p)2.

In some embodiments, the conjugated moiety is a lipid moiety. In someembodiments, any of the siNAs disclosed herein are attached to aconjugated moiety that is a lipid moiety. Examples of lipid moietiesinclude, but are not limited to, a cholesterol mnoiety, a thioether,e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1-di-O-hexadecyl-rac-glycero-S-H-phosphonate, a polyamine or apolyethylene glycol chain, adamantane acetic acid, a palmityl moiety, oran octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

In some embodiments, the conjugated moiety is an active drug substance.In some embodiments, any of the siNAs disclosed herein are attached to aconjugated moiety that is an active drug substance. Examples of activedrug substances include, but are not limited to, aspirin, warfarin,phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(5)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic.

5′-Stabilized End Cap

Further disclosed herein are siNA molecules comprising a 5′-stabilizedend cap. As used herein the terms “5′-stabilized end cap” and “5′ endcap” are used interchangeably. In some embodiments, a 2′-O-methylnucleotide in any of sense strands or first nucleotide sequencesdisclosed herein is replaced with a nucleotide containing a5′-stabilized end cap. In some embodiments, a 2′-O-methyl nucleotide inany of antisense strands or second nucleotide sequences disclosed hereinis replaced with a nucleotide containing a 5′-stabilized end cap. Insome embodiments, a 2′-O-methyl nucleotide in any of sense strands orfirst nucleotide sequences disclosed herein is further modified tocontain a 5′-stabilized end cap. In some embodiments, a 2′-O-methylnucleotide in any of antisense strands or second nucleotide sequencesdisclosed herein is further modified to contain a 5′-stabilized end cap.

In some embodiments, the 5′-stabilized end cap is a 5′ phosphate mimic.In some embodiments, the 5′-stabilized end cap is a modified 5′phosphate mimic. In some embodiments, the modified 5′ phosphate is achemically modified 5′ phosphate. In some embodiments, the 5′-stabilizedend cap is a 5′-vinyl phosphonate. In some embodiments, the 5′-vinylphosphonate is a 5′-(E)-vinyl phosphonate or 5′-(Z)-vinyl phosphonate.In some embodiments, the 5′-vinyl phosphonate is a deuterated vinylphosphonate. In some embodiments, the deuterated vinyl phosphonate is amono-deuterated vinyl phosphonate. In some embodiments, the deuteratedvinyl phosphonate is a di-deuterated vinyl phosphonate. In someembodiments, the 5′-stabilized end cap is a phosphate mimic. Examples ofphosphate mimics are disclosed in Parmar et al., 2018, J Med Chem,61(3):734-744, International Publication Nos. WO2018/045317 andWO2018/044350, and U.S. Pat. No. 10,087,210, each of which isincorporated by reference in its entirety.

In some embodiments, any of the siNA molecules, sense strands, firstnucleotide sequences, antisense strands, or second nucleotide sequencesdisclosed herein comprise a 5′-stabilized end cap of Formula (Ia):

wherein R¹ is H, a nucleobase, aryl, or heteroaryl R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is H; or R² and R²⁰ together form a 3- to 7-membered carbocyclicring substituted with —(CR²¹R²²)_(n)-Z or —(C₂-C₆ alkenylene)-Z; n is 1,2, 3, or 4; Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,—OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃), —P(O)(OH)(OCD₃),—SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴, —NR²³SO₂R²⁴; either R²¹and R²² are independently hydrogen or C₁-C₆ alkyl, or R²¹ and R²²together form an oxo group; R²³ is hydrogen or C₁-C₆ alkyl; R²⁴ is—SO₂R²⁵ or —C(O)R²⁵; or R²³ and R²⁴ together with the nitrogen to whichthey are attached form a substituted or unsubstituted heterocyclic ring;R²⁵ is C₁-C₆ alkyl; and m is 1, 2, 3, or 4. In some embodiments, R¹ isan aryl. In some embodiments, the aryl is a phenyl.

In some embodiments, any of the siNA molecules, sense strands, firstnucleotide sequences, antisense strands, or second nucleotide sequencesdisclosed herein comprise a 5′-stabilized end cap of Formula (Ib):

wherein R¹ is H, a nucleobase, aryl, or heteroaryl; R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is H; or R² and R²⁰ together form a 3- to 7-membered carbocyclicring substituted with —(CR²¹R²²)—Z or —(C₂-C₆ alkenylene)-Z; n is 1, 2,3, or 4; Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,—OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃), —P(O)(OH)(OCD₃),—SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴, —NR²³SO₂R²⁴; either R²¹and R²² are independently hydrogen or C₁-C₆ alkyl, or R²¹ and R²²together form an oxo group; R²³ is hydrogen or C₁-C₆ alkyl; R²⁴ is—SO₂R²⁵ or —C(O)R²⁵; or R²³ and R²⁴ together with the nitrogen to whichthey are attached form a substituted or unsubstituted heterocyclic ring;R²⁵ is C₁-C₆ alkyl; and m is 1, 2, 3, or 4. In some embodiments, R¹ isan aryl. In some embodiments, the aryl is a phenyl.

In some embodiments, any of the siNA molecules, sense strands, firstnucleotide sequences, antisense strands, or second nucleotide sequencesdisclosed herein comprise a 5′-stabilized end cap of Formula (Ic):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H,

-   -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)—Z, or —(C₂-C₆ alkenylene)-Z andR²⁰ is hydrogen; or R² and R²⁰ together form a 3- to 7-memberedcarbocyclic ring substituted with —(CR²¹R²²)r-Z or —(C₂-C₆alkenylene)-Z; n is 1, 2, 3, or 4; Z is —ONR²³R²⁴,—OP(O)OH(CH₂)_(m)CO₂R²³, —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂,—P(O)(OH)(OCH₃), —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²¹,—NR²³R²⁴, or —NR²³SO₂R²⁴; R²¹ and R²² either are independently hydrogenor C₁-C₆ alkyl, or R²¹ and R²² together form an oxo group; R²³ ishydrogen or C₁-C₆ alkyl; R²⁴ is —SO₂R^(2S) or —C(O)R²⁵; or R²³ and R²⁴together with the nitrogen to which they are attached form a substitutedor unsubstituted heterocyclic ring; R²⁵ is C₁-C₆ alkyl; and m is 1, 2,3, or 4. In some embodiments, R¹ is an aryl. In some embodiments, thearyl is a phenyl.

In some embodiments, any of the siNA molecules, sense strands, firstnucleotide sequences, antisense strands, or second nucleotide sequencesdisclosed herein comprise a 5′-stabilized end cap of Formula (IIa):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H, R² is

R⁹ is —SO₂CH₃ or —COCH₃,

is a double or single bond, R¹⁰═—CH₂PO₃H or —NHCH₃, R¹¹ is —CH₂— or—CO—, and R¹² is H and R¹³ is CH₃ or R¹² and R¹³ together form—CH₂CH₂CH₂—. In some embodiments, R¹ is an aryl. In some embodiments,the aryl is a phenyl.

In some embodiments, any of the siNA molecules, sense strands, firstnucleotide sequences, antisense strands, or second nucleotide sequencesdisclosed herein comprise a 5′-stabilized end cap of Formula (IIb):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H, R² is

R⁹ is —SO₂CH₃ or —COCH₃,

is a double or single bond, R¹⁰═—CH₂PO₃H or —NHCH₃, R¹¹ is —CH₂— or—CO—, and R¹² is H and R¹³ is CH₃ or R¹² and R¹³ together form—CH₂CH₂CH₂—. In some embodiments, R¹ is an aryl. In some embodiments,the aryl is a phenyl.

In some embodiments, any of the siNA molecules, sense strands, firstnucleotide sequences, antisense strands, or second nucleotide sequencesdisclosed herein comprise a 5′-stabilized end cap of Formula (II):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H, L is —CH₂—, —CH═CH—,—CO—, or —CH₂CH₂—, and A is —ONHCOCH₃, —ONHSO₂CH₃, —PO₃H,—OP(SOH)CH₂CO₂H, —SO₂CH₂PO₃H, —SO₂NHCH₃, —NHSO₂CH₃, or —N(SO₂CH₂CH₂CH₂).In some embodiments, R¹ is an aryl. In some embodiments, the aryl is aphenyl.

In some embodiments, any of the siNA molecules, sense strands, firstnucleotide sequences, antisense strands, or second nucleotide sequencesdisclosed herein comprise a 5′-stabilized end cap selected from Examples5-11, 33-35, 38, 39, 43, and 49-53 5′ end cap monomers.

Further disclosed herein are siNA molecules comprising (a) a5′-stabilized end cap of Formula (Ia):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H; R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is H; or R² and R²⁰ together form a 3- to 7-membered carbocyclicring substituted with —(CR²¹R²²)-Z or —(C₂-C₆ alkenylene)-Z; n is 1, 2,3, or 4; Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,—OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃), —P(O)(OH)(OCD₃),—SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴, —NR²³SO₂R²⁴; either R²¹and R²² are independently hydrogen or C₁-C₆ alkyl, or R²¹ and R²²together form an oxo group; R²³ is hydrogen or C₁-C₆ alkyl; R²⁴ is—SO₂R²¹ or —C(O)R^(2S); or R²³ and R²⁴ together with the nitrogen towhich they are attached form a substituted or unsubstituted heterocyclicring; R²¹ is C₁-C₆ alkyl; and m is 1, 2, 3, or 4; and (b) a shortinterfering nucleic acid (siNA), wherein the 5′-stabilized end cap isconjugated to the siNA. In some embodiments, R¹ is an aryl. In someembodiments, the aryl is a phenyl.

Further disclosed herein are siNA molecules comprising (a) a5′-stabilized end cap of Formula (Ib):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H; R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is H; or R² and R²⁰ together form a 3- to 7-membered carbocyclicring substituted with —(CR²¹R²²)_(n)-Z or —(C₂-C₆ alkenylene)-Z; n is 1,2, 3, or 4; Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,—OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃), —P(O)(OH)(OCD₃),—SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴, —NR²³SO₂R²⁴; either R²¹and R²² are independently hydrogen or C₁-C₆ alkyl, or R²¹ and R²²together form an oxo group; R²³ is hydrogen or C₁-C₆ alkyl; R²⁴ is—SO₂R²⁵ or —C(O)R²⁵; or R²³ and R²⁴ together with the nitrogen to whichthey are attached form a substituted or unsubstituted heterocyclic ring;R²⁵ is C₁-C₆ alkyl; and m is 1, 2, 3, or 4; and (b) a short interferingnucleic acid (siNA), wherein the 5′-stabilized end cap is conjugated tothe siNA. In some embodiments, R¹ is an aryl. In some embodiments, thearyl is a phenyl.

Further disclosed herein are siNA molecules comprising (a) a5′-stabilized end cap of Formula (Ic):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H, R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)—Z, or —(C₂-C₆ alkenylene)-Z andR²⁰ is hydrogen; or R² and R²⁰ together form a 3- to 7-memberedcarbocyclic ring substituted with —(CR²¹R²²)_(n)-Z or —(C₂-C₆alkenylene)-Z; n is 1, 2, 3, or 4; Z is —ONR²¹R²⁴,—OP(O)OH(CH₂)_(m)CO₂R²³, —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂,—P(O)(OH)(OCH₃), —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²¹,—NR²³R²⁴, or —NR²³SO₂R²⁴; R²¹ and R²² either are independently hydrogenor C₁-C₆ alkyl, or R² and R²² together form an oxo group; R²³ ishydrogen or C₁-C₆ alkyl; R²⁴ is —SO₂R^(2S) or —C(O)R²⁵; or R²³ and R²⁴together with the nitrogen to which they are attached form a substitutedor unsubstituted heterocyclic ring; R²⁵ is C₁-C₆ alkyl; and m is 1, 2,3, or 4; and (b) a short interfering nucleic acid (siNA), wherein the5′-stabilized end cap is conjugated to the siNA. In some embodiments, R¹is an aryl. In some embodiments, the aryl is a phenyl.

In some embodiments, a siNA molecule comprises (a) a 5′-stabilized endcap of

Formula (Ha):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H, R² is

R⁹ is —SO₂CH₃ or —COCH₃, wherein

is a double or single bond, R¹⁰═—CH₂PO₃H or —NHCH₃, R¹¹ is —CH₂— or—CO—, and R¹² is H and R³ is CH₃ or R² and R¹³ together form—CH₂CH₂CH₂—; and (b) a short interfering nucleic acid (siNA), whereinthe 5′-stabilized end cap is conjugated to the siNA. In someembodiments, R¹ is an aryl. In some embodiments, the aryl is a phenyl.

In some embodiments, a siNA molecule comprises (a) a 5′-stabilized endcap of Formula (IIb)

wherein R¹ is a nucleobase, aryl, heteroaryl, or H, R² is

R⁹ is —SO₂CH₃ or —COCH₃, wherein is a double or single bond,R¹⁰═—CH₂PO₃H or —NHCH₃, R¹¹ is —CH₂— or —CO—, and R¹² is H and R¹³ isCH₃ or R¹² and R¹³ together form —CH₂CH₂CH₂—; and (b) a shortinterfering nucleic acid (siNA), wherein the 5′-stabilized end cap isconjugated to the siNA. In some embodiments, R¹ is an aryl. In someembodiments, the aryl is a phenyl.

In some embodiments, a siNA molecule comprises (a) a 5′-stabilized endcap of Formula (III):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H, L is ˜CH₂—, —CH═CH—,—CO—, or —CH₂CH₂—, and A is —ONHCOCH₃, —ONHSO₂CH₃, —PO₃H,—OP(SOH)CH₂CO₂H, —SO₂CH₂PO₃H, —SO₂NHCH₃, —NHSO₂CH₃, or —N(SO₂CH₂CH₂CH₂);and (b) a short interfering nucleic acid (siNA), wherein the5′-stabilized end cap is conjugated to the siNA. In some embodiments, R¹is an aryl. In some embodiments, the aryl is phenyl.

In some embodiments, any of the siNA molecules disclosed herein comprisea 5′-stabilized end cap selected from the group consisting of Formula(1) to Formula (15), Formula (9X) to Formula (12X), and Formula (9Y) toFormula (12Y):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H. In some embodiments,R¹ is an aryl. In some embodiments, the aryl is a phenyl.

In some embodiments, any of the siNA molecules disclosed herein comprisea 5′-stabilized end cap selected from the group consisting of Formulas(1A)-(15A), Formulas (9B)-(12B), Formulas (9AX)-(12AX), Formulas(9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas (9BY)-(12BY):

In some embodiments, any of the siNA molecules disclosed herein comprisea 5′-stabilized end cap selected from the group consisting of Formula(21) to Formula (35):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H. In some embodiments,R¹ is an aryl. In some embodiments, the aryl is a phenyl.

In some embodiments, any of the siNA molecules disclosed herein comprisea 5′-stabilized end cap selected from the group consisting of Formulas(21A)-(35A), Formulas (29B)-(32B), Formulas (29AX)-(32AX), Formulas(29AY)-(32AY), Formulas 29BX 32BX and Formulas 29BY 32BY):

In some embodiments, the 5′-stabilized end cap is attached to the 5′ endof the antisense strand. In some embodiments, the 5′-stabilized end capis attached to the 5′ end of the antisense strand via 1, 2, 3, 4, or 5or more linkers. In some embodiments, the one or more linkers areindependently selected from the group consisting of a phosphodiester (por po) linker, phosphorothioate (ps) linker (ps), phosphoramidite (HEG)linker, triethylene glycol (TEG) linker, and/or phosphorodithioatelinker. In some embodiments, the one or more linkers are independentlyselected from the group consisting of p-(PS)2, (PS)2-p-TEG-p,(PS)2-p-HEG-p, and (PS)2-p-(HEG-p)2.

Linker

In some embodiments, any of the siRNAs, sense strands, first nucleotidesequences, antisense strands, and/or second nucleotide sequencesdisclosed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or more internucleoside linkers. Insome embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more internucleosidelinkers are independently selected from the group consisting of aphosphodiester (p or po) linker, phosphorothioate (ps) linker, orphosphorodithioate linker.

In some embodiments, any of the siRNAs, sense strands, first nucleotidesequences, antisense strands, and/or second nucleotide sequencesdisclosed herein further comprise 1, 2, 3, 4 or more linkers that attacha conjugated moiety, phosphorylation blocker, and/or 5′ end cap to thesiRNA, sense strand, first nucleotide sequence, antisense strand, and/orsecond nucleotide sequences. In some embodiments, the 1, 2, 3, 4 or morelinkers are independently selected from the group consisting of aphosphodiester (p or po) linker, phosphorothioate (ps) linker,phosphoramidite (HEG) linker, triethylene glycol (TEG) linker, and/orphosphorodithioate linker. In some embodiments, the one or more linkersare independently selected from the group consisting of p-(PS)2,(PS)2-p-TEG-p, (PS)2-p-HEG-p, and (PS)2-p-(HEG-p)2.

Target Gene

Without wishing to be bound by theory, upon entry into a cell, any ofthe ds-siNA molecules disclosed herein may interact with proteins in thecell to form a RNA-Induced Silencing Complex (RISC). Once the ds-siNA ispart of the RISC, the ds-siNA may be unwound to form a single-strandedsiNA (ss-siNA). The ss-siNA may comprise the antisense strand of theds-siNA. The antisense strand may bind to a complementary messenger RNA(mRNA), which results in silencing of the gene that encodes the mRNA.

The target gene may be any gene in a cell. In some embodiments, thetarget gene is a viral gene. In some embodiments, the viral gene is froma DNA virus. In some embodiments, the DNA virus is a double-stranded DNA(dsDNA) virus. In some embodiments, the dsDNA virus is a hepadnavirus.In some embodiments, the hepadnavirus is a hepatitis B virus (HBV). Insome embodiments, the HBV is selected from HBV genotypes A-J.

In some embodiments, the target gene is selected from the S gene or Xgene of the HBV. In some embodiments, the HBV has a genome sequenceshown in the nucleotide sequence of SEQ ID NO: 410, which corresponds tothe nucleotide sequence of GenBank Accession No. U95551.1, which isincorporated by reference in its entirety.

An exemplary HBV genome sequence is shown in SEQ ID NO: 596,corresponding to Genbank Accession No. KC315400.1, which is incorporatedby reference in its entirety. Nucleotides 2307. . 3215, 1. . 1623 of SEQID NO: 596 correspond to the polymerase/RT gene sequence, which encodesfor the polymerase protein. Nucleotides 2848. . 3215,1. . 835 of SEQ IDNO: 596 correspond to the PreS1/S2/S gene sequence, which encodes forthe large S protein. Nucleotides 3205. . 3215,1. . 835 of SEQ ID NO: 596correspond to the PreS2/S gene sequence, which encodes for the middle Sprotein. Nucleotides 155. . 835 of SEQ ID NO: 596 correspond to the Sgene sequence, which encodes the small S protein. Nucleotides 1374. .1838 of SEQ ID NO: 596 correspond to the X gene sequence, which encodesthe X protein. Nucleotides 1814. . 2452 of SEQ ID NO: 596 correspond tothe PreC/C gene sequence, which encodes the precore/core protein.Nucleotides 1901. . 2452 of SEQ ID NO: 596 correspond to the C genesequence, which encodes the core protein. The HBV genome furthercomprises viral regulatory elements, such as viral promoters (preS2,preS1, Core, and X) and enhancer elements (ENH1 and ENH2). Nucleotides1624. . 1771 of SEQ ID NO: 596 correspond to ENH2. Nucleotides 1742. .1849 of SEQ ID NO: 596 correspond to the Core promoter. Nucleotides 1818. . . 3215,1. . 1930 of SEQ ID NO: 596 correspond to the pregenomic RNA(pgRNA), which encodes the core and polymerase proteins.

In some embodiments, the ASO is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% complementary or hybridizes to a viral target RNAsequence that begins in an X region of HBV or in an S region of HBV. Theviral target may, e.g., begin at the 5′-end of target-site in acc.KC315400.1 (genotype B, “gt B”), or in any one of genotypes A, C, or D.The skilled person would understand the HBV position, e.g., as describedin Wing-Kin Sung, et al., Nature Genetics 44:765 (2012). In someembodiments, the S region is defined as from the beginning of small Sprotein (in genotype B KC315400.1 isolate, position #155) to beforebeginning of X protein (in genotype B KC315400.1 isolate, position#1373). In some embodiments, the X region is defined as from thebeginning X protein (in genotype B KC315400.1 isolate, position #1374)to end of DR2 site (in genotype B KC315400.1 isolate, position #1603).

In some embodiments, the second nucleotide sequence is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to22, 17 to 21, or 19 to 21 nucleotides within positions 200-720 or1100-1700 of SEQ ID NO: 410. In some embodiments, the second nucleotidesequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% complementary to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21,17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides withinpositions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or1550-1630 of SEQ ID NO: 410. In some embodiments, the second nucleotidesequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% complementary to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21,17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides withinpositions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700,1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO: 410. In someembodiments, the second nucleotide sequence is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30, 15 to25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to21,or 19 to 21 nucleotides starting at position 203, 206, 254, 305, 375,409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263,1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO: 410.

In some embodiments, the first nucleotide is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a nucleotide regionwithin SEQ ID NO: 410, with the exception that the thymines (Ts) in SEQID NO: 410 are replaced with uracil (U). In some embodiments, the firstnucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% identical to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotideswithin positions 200-720 or 1100-1700 of SEQ ID NO: 410. In someembodiments, the first nucleotide sequence is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30, 15 to 25,15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21,or19 to 21 nucleotides within positions 200-280, 300-445, 460-510,650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410. In someembodiments, the first nucleotide sequence is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30, 15 to 25,15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or19 to 21 nucleotides within positions 200-230, 250-280, 300-330,370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or1570-1610 of SEQ ID NO: 410. In some embodiments, the first nucleotidesequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% identical to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides starting atposition 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466,467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581,1583, or 1584 of SEQ ID NO: 410.

In some embodiments, the target gene is involved in liver metabolism. Insome embodiments, the target gene is an inhibitor of the electrontransport chain. In some embodiments, the target gene encodes the MCJprotein (MCJ/DnaJC15 or Methylation-Controlled J protein). In someembodiments, the MCJ protein is encoded by the mRNA sequence of SEQ IDNO: 411, which corresponds to the nucleotide sequence of GenBankAccession No. NM_013238.3, which is incorporated by reference in itsentirety.

In some embodiments, the target gene is TAZ. In some embodiments, TAZcomprises the nucleotide sequence of SEQ ID NO: 412, which correspondsto the nucleotide sequence of GenBank Accession No. NM_000116.5, whichis incorporated by reference in its entirety.

In some embodiments, the target gene is angiopoietin like 3 (ANGPTL3).In some embodiments, ANGPTL3 comprises the nucleotide sequence of SEQ IDNO: 413, which corresponds to the nucleotide sequence of GenBankAccession No. NM_014495.4, which is incorporated by reference in itsentirety.

In some embodiments, the target gene is diacylglycerol acyltransferase 2(DGAT2). In some embodiments, DGAT2 comprises the nucleotide sequence ofSEQ ID NO: 414, which corresponds to the nucleotide sequence of GenBankAccession No. NM_001253891.1, which is incorporated by reference in itsentirety.

Compositions

As indicated above, the present disclosure provides compositionscomprising any of the siNA molecules, sense strands, antisense strands,first nucleotide sequences, or second nucleotide sequences describedherein. The compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 or more siNA molecules described herein. The compositions maycomprise a first nucleotide sequence comprising a nucleotide sequence ofany one SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments, thecomposition comprises a second nucleotide sequence comprising anucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and261-306. In some embodiments, the composition comprises a sense strandcomprising a nucleotide sequence of any one of SEQ ID NOs: 307-362 and415-444. In some embodiments, the composition comprises an antisensestrand comprising a nucleotide sequence of any one of SEQ ID NOs:363-409, 445-533, and 536-539.

Alternatively, the compositions may comprise (a) a phosphorylationblocker; and (b) a short interfering nucleic acid (siNA). In someembodiments, the phosphorylation blocker is any of the phosphorylationblockers disclosed herein. In some embodiments, the siNA is any of thesiNAs disclosed herein. In some embodiments, the siNA comprises any ofthe sense strands, antisense strands, first nucleotide sequences, orsecond nucleotide sequences described herein. In some embodiments, thesiNA comprises any of the sense strands, antisense strands, firstnucleotide sequences, or second nucleotide sequences described herein.In some embodiments, the siNA comprises one or more modifiednucleotides. In some embodiments, the one or more modified nucleotidesare independently selected from a 2′-fluoro nucleotide and a 2′-O-methylnucleotide. In some embodiments, the 2′-fluoro nucleotide or the2′-O-methyl nucleotide is independently selected from any of the2′-fluoro or 2′-O-methyl nucleotide mimics disclosed herein. In someembodiments, the siNA comprises a nucleotide sequence comprising any ofthe modification patterns disclosed herein.

In some embodiments, the composition comprises (a) a conjugated moiety;and (b) a short interfering nucleic acid (siNA). In some embodiments,the conjugated moiety is any of the galactosamines disclosed herein. Insome embodiments, the siNA is any of the siNAs disclosed herein. In someembodiments, the siNA comprises any of the sense strands, antisensestrands, first nucleotide sequences, or second nucleotide sequencesdescribed herein. In some embodiments, the siNA comprises any of thesense strands, antisense strands, first nucleotide sequences, or secondnucleotide sequences described herein. In some embodiments, the siNAcomprises one or more modified nucleotides. In some embodiments, the oneor more modified nucleotides are independently selected from a 2′-fluoronucleotide and a 2′-O-methyl nucleotide. In some embodiments, the2′-fluoro nucleotide or the 2′-O-methyl nucleotide is independentlyselected from any of the 2′-fluoro or 2′-O-methyl nucleotide mimicsdisclosed herein. In some embodiments, the siNA comprises a nucleotidesequence comprising any of the modification patterns disclosed herein.

In some embodiments, the composition comprises (a) a 5′-stabilized endcap; and (b) a short interfering nucleic acid (siNA). In someembodiments, the 5′-stabilized end cap is any of the 5-stabilized endcaps disclosed herein. In some embodiments, the siNA is any of the siNAsdisclosed herein. In some embodiments, the siNA comprises any of thesense strands, antisense strands, first nucleotide sequences, or secondnucleotide sequences described herein. In some embodiments, the siNAcomprises one or more modified nucleotides. In some embodiments, the oneor more modified nucleotides are independently selected from a 2′-fluoronucleotide and a 2′-O-methyl nucleotide. In some embodiments, the2′-fluoro nucleotide or the 2′-O-methyl nucleotide is independentlyselected from any of the 2′-fluoro or 2′-O-methyl nucleotide mimicsdisclosed herein. In some embodiments, the siNA comprises a nucleotidesequence comprising any of the modification patterns disclosed herein.

In some embodiments, the composition comprises (a) at least onephosphorylation blocker, conjugated moiety, or 5′-stabilized end cap;and (b) a short interfering nucleic acid (siNA). In some embodiments,the phosphorylation blocker is any of the phosphorylation blockersdisclosed herein. In some embodiments, the conjugated moiety is any ofthe galactosamines disclosed herein. In some embodiments, the5′-stabilized end cap is any of the 5-stabilized end caps disclosedherein. In some embodiments, the siNA is any of the siNAs disclosedherein. In some embodiments, the siNA comprises any of the sensestrands, antisense strands, first nucleotide sequences, or secondnucleotide sequences described herein. In some embodiments, the siNAcomprises one or more modified nucleotides. In some embodiments, the oneor more modified nucleotides are independently selected from a 2′-fluoronucleotide and a 2′-O-methyl nucleotide. In some embodiments, the2′-fluoro nucleotide or the 2′-O-methyl nucleotide is independentlyselected from any of the 2′-fluoro or 2′-O-methyl nucleotide mimicsdisclosed herein. In some embodiments, the siNA comprises a nucleotidesequence comprising any of the modification patterns disclosed herein.

The composition may be a pharmaceutical composition. In someembodiments, the pharmaceutical composition comprises an amount of oneor more of the siNA molecules described herein formulated with one ormore pharmaceutically acceptable carriers (additives) and/or diluents.The pharmaceutical compositions may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a siNA of thepresent disclosure which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present disclosure include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound (e.g., siNA molecule) which produces atherapeutic effect. Generally, out of one hundred percent, this amountwill range from about 0.1 percent to about ninety-nine percent of activeingredient, preferably from about 5 percent to about 70 percent, mostpreferably from about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present disclosurecomprises an excipient selected from the group consisting ofcyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bileacids, and polymeric carriers, e.g., polyesters and polyanhydrides; anda compound (e.g., siNA molecule) of the present disclosure. In certainembodiments, an aforementioned formulation renders orally bioavailable acompound (e.g., siNA molecule) of the present disclosure.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound (e.g., siNA molecule) of thepresent disclosure with the carrier and, optionally, one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association a compound (e.g.,siNA molecule) of the present disclosure with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Formulations of the disclosure suitable for oral administration may bein the form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound (e.g., siNAmolecule) of the present disclosure as an active ingredient. A compound(e.g., siNA molecule) of the present disclosure may also be administeredas a bolus, electuary or paste.

In solid dosage forms of the disclosure for oral administration(capsules, tablets, pills, dragées, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof, (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose.

In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-shelledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present disclosure, such as dragées, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried.

They may be sterilized by, for example, filtration through abacteria-retaining filter, or by incorporating sterilizing agents in theform of sterile solid compositions which can be dissolved in sterilewater, or some other sterile injectable medium immediately before use.These compositions may also optionally contain opacifying agents and maybe of a composition that they release the active ingredient(s) only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeingredient can also be in micro-encapsulated form, if appropriate, withone or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds (e.g., siNAmolecules) of the disclosure include pharmaceutically acceptableemulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (Iparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds (e.g., siNA molecules),may contain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the disclosure forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds (e.g., siNAmolecules) of the disclosure with one or more suitable nonirritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax or a salicylate, and which issolid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound (e.g., siNA molecule).

Formulations of the present disclosure which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compound(e.g., siNA molecule) of this disclosure include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound (e.g., siNA molecule) may be mixed understerile conditions with a pharmaceutically acceptable carrier, and withany preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound (e.g., siNA molecule) of this disclosure, excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound (e.g., siNAmolecule) of this disclosure, excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound (e.g., siNA molecule) of the present disclosureto the body. Such dosage forms can be made by dissolving or dispersingthe compound (e.g., siNA molecule) in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound (e.g.,siNA molecule) across the skin. The rate of such flux can be controlledby either providing a rate controlling membrane or dispersing thecompound (e.g., siNA molecule) in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this disclosure suitable for parenteraladministration comprise one or more compounds (e.g., siNA molecules) ofthe disclosure in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain sugars, alcohols,antioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds (e.g., siNA molecules) in biodegradable polymerssuch as polylactide-polyglycolide. Depending on the ratio of drug topolymer, and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds (e.g., siNA molecules) of the present disclosure areadministered as pharmaceuticals, to humans and animals, they can begiven per se or as a pharmaceutical composition containing, for example,0.1 to 99% (more preferably, 10 to 30%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of Treatment and Administration

The siNA molecules of the present disclosure may be used to treat adisease in a subject in need thereof. In some embodiments, a method oftreating a disease in a subject in need thereof comprises administeringto the subject any of the siNA molecules disclosed herein. In someembodiments, a method of treating a disease in a subject in need thereofcomprises administering to the subject any of the compositions disclosedherein.

The preparations (e.g., siNA molecules or compositions) of the presentdisclosure may be given orally, parenterally, topically, or rectally.They are of course given in forms suitable for each administrationroute. For example, they are administered in tablets or capsule form,administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories. Oral administrations arepreferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds (e.g.,siNA molecules) of the present disclosure, which may be used in asuitable hydrated form, and/or the pharmaceutical compositions of thepresent disclosure, are formulated into pharmaceutically-acceptabledosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this disclosure may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound (e.g., siNA molecule)of the present disclosure employed, or the ester, salt or amide thereof,the route of administration, the time of administration, the rate ofexcretion or metabolism of the particular compound being employed, therate and extent of absorption, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds (e.g., siNA molecules) of the disclosureemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound (e.g., siNA molecule) ofthe disclosure is the amount of the compound that is the lowest doseeffective to produce a therapeutic effect. Such an effective dosegenerally depends upon the factors described above. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. In some embodiments, the compoundis administered at a dose equal to or greater than 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15,0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27,0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7, 0.75,0.8, 0.85, 0.9, 0.95, or 1 mg/kg. In some embodiments, the compound isadministered at a dose equal to or less than 200, 190, 180, 170, 160,150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50,45, 40, 35, 30, 25, 20, or 15 mg/kg. In some embodiments, the totaldaily dose of the compound is equal to or greater than 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, or 100 mg.

When the compounds (e.g., siNA molecules) described herein areco-administered with another, the effective amount may be less than whenthe compound is used alone.

If desired, the effective daily dose of the active compound (e.g., siNAmolecule) may be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms. Preferred dosing is oneadministration per day. In some embodiments, the compound isadministered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 21 times a week. In some embodiments, thecompound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 times a month. In someembodiments, the compound is administered once every 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In someembodiments, the compound is administered once every 1, 2, 3, 4, 5, 6,7, or 8 weeks.

Diseases

The siNA molecules and compositions described herein may be administeredto a subject to treat a disease. Further disclosed herein are uses ofany of the siNA molecules or compositions disclosed herein in themanufacture of a medicament for treating a disease.

In some embodiments, the disease is a viral disease. In someembodiments, the viral disease is caused by a DNA virus. In someembodiments, the DNA virus is a double stranded DNA (dsDNA virus). Insome embodiments, the dsDNA virus is a hepadnavirus. In someembodiments, the hepadnavirus is a hepatitis B virus (HBV).

In some embodiments, the disease is a liver disease. In someembodiments, the liver disease is nonalcoholic fatty liver disease(NAFLD). In some embodiments, the NAFLD is nonalcoholic steatohepatitis(NASH). In some embodiments, the liver disease is hepatocellularcarcinoma (HCC).

Administration of siNA

Administration of any of the siNAs disclosed herein may be conducted bymethods known in the art. In some embodiments, the siNA is administeredby subcutaneous (SC) or intravenous (IV) delivery. The preparations(e.g., siNAs or compositions) of the present disclosure may be givenorally, parenterally, topically, or rectally. They are of course givenin forms suitable for each administration route. For example, they areadministered in tablets or capsule form, administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. In some embodiments, subcutaneous administration ispreferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds (e.g.,siNAs) of the present disclosure, which may be used in a suitablehydrated form, and/or the pharmaceutical compositions of the presentdisclosure, are formulated into pharmaceutically-acceptable dosage formsby conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this disclosure may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound (e.g., siNA) of thepresent disclosure employed, or the ester, salt or amide thereof, theroute of administration, the time of administration, the rate ofexcretion or metabolism of the particular compound being employed, therate and extent of absorption, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds (e.g., siNAs) of the disclosure employed inthe pharmaceutical composition at levels lower than that required inorder to achieve the desired therapeutic effect and gradually increasethe dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound (e.g., siNA) of thedisclosure is the amount of the compound that is the lowest doseeffective to produce a therapeutic effect. Such an effective dosegenerally depends upon the factors described above. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. In some embodiments, the compoundis administered at about 1 mg/kg to about 40 mg/kg, about 1 mg/kg toabout 30 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about15 mg/kg, or 1 mg/kg to about 10 mg/kg. In some embodiments, thecompound is administered at a dose equal to or greater than 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14,0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26,0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7,0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg/kg. In some embodiments, thecompound is administered at a dose equal to or greater than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 mg/kg. In some embodiments, the compoundis administered at a dose equal to or less than 200, 190, 180, 170, 160,150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50,45, 40, 35, 30, 25, 20, or 15 mg/kg. In some embodiments, the totaldaily dose of the compound is equal to or greater than 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, or 100 mg.

If desired, the effective daily dose of the active compound (e.g., siNA)may be administered as two, three, four, five, six, seven, eight, nine,ten or more doses or sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. In someembodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 times. Preferred dosing is oneadministration per day. In some embodiments, the compound isadministered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 21 times a week. In some embodiments, thecompound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 times a month. In someembodiments, the compound is administered once every 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In someembodiments, the compound is administered every 3 days. In someembodiments, the compound is administered once every 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks. In some embodiments, thecompound is administered every month. In some embodiments, the compoundis administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 months. In some embodiments, the compound is administered atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 timesover a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, or 70 days. In some embodiments, the compound is administered atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 timesover a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, or 53 weeks. In some embodiments, the compound is administeredat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53times over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, or 53 months. In some embodiments, the compound isadministered at least once a week for a period of at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In someembodiments, the compound is administered at least once a week for aperiod of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,or 70 months. In some embodiments, the compound is administered at leasttwice a week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, or 70 weeks. In some embodiments, the compound isadministered at least twice a week for a period of at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months. In someembodiments, the compound is administered at least once every two weeksfor a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,or 70 weeks. In some embodiments, the compound is administered at leastonce every two weeks for a period of at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, or 70 months. In some embodiments, the compoundis administered at least once every four weeks for a period of at least4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In someembodiments, the compound is administered at least once every four weeksfor a period of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or70 months.

In some embodiments, any one of the siNAs or compositions disclosedherein is administered in a particle or viral vector. In someembodiments, the viral vector is a vector of adenovirus,adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplexvirus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, orrhabdovirus. In some embodiments, the viral vector is a recombinantviral vector. In some embodiments, the viral vector is selected fromAAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6,AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13.

The subject of the described methods may be a mammal, and it includeshumans and non-human mammals. In some embodiments, the subject is ahuman, such as an adult human.

Some embodiments include a method for treating an HBV virus in a subjectinfected with the virus comprising administering a therapeuticallyeffective amount of one or more siNA of the present disclosure or acomposition of the present disclosure to the subject in need thereofthereby reducing the viral load of the virus in the subject and/orreducing a level of a virus antigen in the subject. The siNA may becomplementary or hybridize to a portion of the target RNA in the virus,e.g., an X region and/or an S region of HBV.

Combination Therapies

Any of the methods disclosed herein may further comprise administeringto the subject an additional HBV treatment agent. Any of thecompositions disclosed herein may further comprise an additional HBVtreatment agent. In some embodiments, the additional HBV treatment agentis selected from a nucleotide analog, nucleoside analog, a capsidassembly modulator (CAM), a recombinant interferon, an entry inhibitor,a small molecule immunomodulator and oligonucleotide therapy. In someembodiments, the additional HBV treatment agent is selected from HBVSTOPS™ ALG-010133, HBV CAM ALG-000184, ASO 1, recombinant interferonalpha 2b, IFN-α, PEG-IFN-α-2a, lamivudine, telbivudine, adefovirdipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovirdisoproxil, NVR3-778, BAY41-4109, JNJ-632, JNJ-3989 (ARO-HBV), RG6004,GSK3228836, REP-2139, REP-2165, AB-729, VIR-2218, RG6346 (DCR-HBVS),JNJ-6379, GLS4, ABI-HO731, JNJ-440, NZ-4, RG7907, EDP-514, AB-423,AB-506, ABI-H03733 and ABI-H2158. In some embodiments, theoligonucleotide therapy is selected from Nucleic Acid Polymers orS-Antigen Transport-inhibiting Oligonucleotide Polymers (NAPs or STOPS),siRNA, and ASO. In some embodiments, the oligonucleotide therapy is anadditional siNA. In some embodiments, the additional siNA is selectedfrom any of ds-siNA-001 to ds-siNA-0178. In some embodiments, theoligonucleotide therapy is an antisense oligonucleotide (ASO). In someembodiments, the ASO is ASO 1. In some embodiments, any of the siNAsdisclosed herein are co-administered with STOPS. Exemplary STOPS aredescribed in International Publication No. WO2020/097342 and U.S.Publication No. 2020/0147124, both of which are incorporated byreference in their entirety. In some embodiments, the STOPS isALG-010133. In some embodiments, any of the siNAs disclosed herein areco-administered with tenofovir. In some embodiments, any of the siNAsdisclosed herein are co-administered with a CAM. Exemplary CAMs aredescribed in Berke et al., Antimicrob Agents Chemother, 2017,61(8):e00560-17, Klumpp, et al., Gastroenterology, 2018,154(3):652-662.e8, International Application Nos. PCT/US2020/017974,PCT/US2020/026116, and PCT/US2020/028349 and U.S. application Ser. Nos.16/789,298, 16/837,515, and 16/849,851, each which is incorporated byreference in its entirety. In some embodiments, the CAM is ALG-000184,ALG-001075, ALG-001024, JNJ-632, BAY41-4109, or NVR3-778. In someembodiments, the siNA and the HBV treatment agent are administeredsimultaneously. In some embodiments, the siNA and the HBV treatmentagent are administered concurrently. In some embodiments, the siNA andthe HBV treatment agent are administered sequentially. In someembodiments, the siNA is administered prior to administering the HBVtreatment agent. In some embodiments, the siNA is administered afteradministering the HBV treatment agent. In some embodiments, the siNA andthe HBV treatment agent are in separate containers. In some embodiments,the siNA and the HBV treatment agent are in the same container.

Any of the methods disclosed herein may further comprise administeringto the subject a liver disease treatment agent. Any of the compositionsdisclosed herein may further comprise a liver disease treatment agent.In some embodiments, the liver disease treatment agent is selected froma peroxisome proliferator-activator receptor (PPAR) agonist, farnesoid Xreceptor (FXR) agonist, lipid-altering agent, and incretin-basedtherapy. In some embodiments, the PPAR agonist is selected from a PPARαagonist, dual PPARα/δ agonist, PPARγ agonist, and dual PPARα/γ agonist.In some embodiments, the dual PPARα agonist is a fibrate. In someembodiments, the PPARα/δ agonist is elafibranor. In some embodiments,the PPARγ agonist is a thiazolidinedione (TZD). In some embodiments, TZDis pioglitazone. In some embodiments, the dual PPARα/γ agonist issaroglitazar. In some embodiments, the FXR agonist is obeticholic acis(OCA). In some embodiments, the lipid-altering agent is aramchol. Insome embodiments, the incretin-based therapy is a glucagon-like peptide1 (GLP-1) receptor agonist or dipeptidyl peptidase 4 (DPP-4) inhibitor.In some embodiments, the GLP-1 receptor agonist is exenatide orliraglutide. In some embodiments, the DPP-4 inhibitor is sitagliptin orvildapliptin. In some embodiments, the siNA and the liver diseasetreatment agent are administered concurrently. In some embodiments, thesiNA and the liver disease treatment agent are administeredsequentially. In some embodiments, the siNA is administered prior toadministering the liver disease treatment agent. In some embodiments,the siNA is administered after administering the liver disease treatmentagent. In some embodiments, the siNA and the liver disease treatmentagent are in separate containers. In some embodiments, the siNA and theliver disease treatment agent are in the same container.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this disclosure belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al., (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise. The terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the disclosure.

The terms “a” and “an” as used herein mean “one or more” and include theplural unless the context is inappropriate.

As used herein, the terms “patient” and “subject” refer to organisms tobe treated by the methods of the present disclosure. Such organisms arepreferably mammals (e.g., marines, simians, equines, bovines, porcinis,canines, felines, and the like), and more preferably humans.

As used herein, the term “effective amount” refers to the amount of acompound (e.g., a siNA of the present disclosure) sufficient to effectbeneficial or desired results. An effective amount can be administeredin one or more administrations, applications, or dosages and is notintended to be limited to a particular formulation or administrationroute.

As used herein, the term “treating” includes any effect, e.g.,lessening, reducing, modulating, ameliorating or eliminating, thatresults in the improvement of the condition, disease, disorder, and thelike, or ameliorating a symptom thereof.

As used herein, the terms “alleviate” and “alleviating” refer toreducing the severity of the condition, such as reducing the severityby, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 95%.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see, for example,Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co.,Easton, PA [1975].

The term “about” as used herein when referring to a measurable value(e.g., weight, time, and dose) is meant to encompass variations, such as±10%, ±5%, ±1%, or f0.1% of the specified value.

As used herein, the term “nucleobase” refers to a nitrogen-containingbiological compound that forms a nucleoside. Examples of nucleobasesinclude, but are not limited to, thymine, uracil, adenine, cytosine,guanine, aryl, heteroaryl, and an analogue or derivative thereof.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present disclosure that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present disclosure that consist essentially of, or consist of,the recited processing steps.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication datesthat may need to be independently confirmed.

EXAMPLES Example 1. siNA Synthesis

This example describes an exemplary method for synthesizing ds-siNAs,such as the siNAs disclosed in Table 6 (as identified by the ds-siNAID).

The 2′-OMe phosphoramidite 5′-O-DMT-deoxy Adenosine (NH-Bz),3′-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5′-O-DMT-deoxyGuanosine (NH-ibu), 3′-0-(2-cyanoethyl-N,N-diisopropyl phosphoramidite,5′-O-DMT-deoxy Cytosine (NH-Bz), 3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite, 5′-O-DMT-Uridine 3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite and solid supports were purchased from Chemgenes Corp.MA.

The 2′-F -5′-O-DMT-(NH-Bz) Adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite, 2′-F -5′-O-DMT-(NH-ibu)-Guanosine,3′-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite,5′-O-DMT-(NH-Bz)-Cytosine, 2′-F-3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite, 5′-O-DMT-Uridine,2′-F-3′-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite and solidsupports were purchased from Thermo Fischer Milwaukee WI, USA.

All the monomers were dried in vacuum desiccator with desiccants (P₂O₅,RT 24 h). The solid supports (CPG) attached to the nucleosides anduniversal supports was obtained from LGC and Chemgenes. The chemicalsand solvents for post synthesis workflow were purchased fromcommercially available sources like VWR/Sigma and used without anypurification or treatment. Solvent (Acetonitrile) and solutions (amiditeand activator) were stored over molecular sieves during synthesis.

The oligonucleotides were synthesized on a DNA/RNA Synthesizers(Expedite 8909 or ABI-394) using standard oligonucleotidephosphoramidite chemistry starting from the 3′ residue of theoligonucleotide preloaded on CPG support. An extended coupling of 0.1Msolution of phosphoramidite in CH₃CN in the presence of5-(ethylthio)-1H-tetrazole activator to a solid bound oligonucleotidefollowed by standard capping, oxidation and deprotection affordedmodified oligonucleotides. The 0.1 M I₂, THF:Pyridine;Water-7:2:1 wasused as oxidizing agent while DDTT ((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione was used as the sulfur-transferagent for the synthesis of oligoribonucleotide phosphorothioates. Thestepwise coupling efficiency of all modified phosphoramidites was morethan 98%.

Reagents Detailed Description Deblock Solution 3% Dichloroacetic acid(DCA) in Dichloromethane (DCM) Amidite Concentration 0.1M in AnhydrousAcetonitrile Activator 0.25M Ethyl-thio-Tetrazole (ETT) Cap-A solutionAcetic anhydride in Pyridine/THF Cap-B Solution 16% 1-Methylimidazole inTHF Oxidizing Solution 0.02M I₂, THF: Pyridine; Water-7:2:1 SulfurizingSolution 0.2M DDTT in Pyridine/Acetonitrile 1:1

Cleavage and Deprotection:

Deprotection and cleavage from the solid support was achieved withmixture of ammonia methylamine (1:1, AMA) for 15 min at 65° C., when theuniversal linker was used, the deprotection was left for 90 min at 65°C. or solid supports were heated with aqueous ammonia (28%) solution at55° C. for 16 h to deprotect the base labile protecting groups.

Quantitation of Crude SiNA or Raw Analysis

Samples were dissolved in deionized water (1.0 mL) and quantitated asfollows: Blanking was first performed with water alone (2 ul) onNanodrop then Oligo sample reading obtained at 260 nm. The crudematerial is dried down and stored at −20° C.

Crude HPLC/LC-MS Analysis

The 0.1 OD of the crude samples were analyzed for crude HPLC and LC-MSanalysis. After Confirming the crude LC-MS data then purification stepwas performed.

HPLC Purification

The unconjugated and GalNac modified oligonucleotides were purified byanion-exchange HPLC. The buffers were 20 mM sodium phosphate in 10%CH₃CN, pH 8.5 (buffer A) and 20 mM sodium phosphate in 10% CH₃CN, 1.0 MNaBr, pH 8.5 (buffer B). Fractions containing full-lengtholigonucleotides were pooled.

Desalting of Purified SiNA

The purified dry siNA was then desalted using Sephadex G-25 M (AmershamBiosciences). The cartridge was conditioned with 10 mL of deionizedwater thrice. Finally, the purified siNA dissolved thoroughly in 2.5 mLRNAse free water was applied to the cartridge with very slow drop wiseelution. The salt free siNA was eluted with 3.5 ml deionized waterdirectly into a screw cap vial.

IEX HPLC and Electrospray LC/MS Analysis

Approximately 0.10 OD of siNA is dissolved in water and then pipetted inspecial vials for IEX-HPLC and LC/MS analysis. Analytical HPLC and ESLC-MS established the integrity of the compounds.

Duplex Preparation:

Single strand oligonucleotides (Sense and Antisense strands) wereannealed (1:1 by molar equivalents, heat 90° C. for 3 min followed byroom temperature, 20 min) to give the duplex ds-siNA. The finalcompounds were analyzed on size exclusion chromatography (SEC).

Example 2. Ds-siNA Activity

This example investigates the activity of the ds-siNAs synthesized inExample 1.

Homo sapiens HepG2.2.15 cells were cultured in Dulbecco's ModifiedEagle's Medium (DMEM) (ATCC 30-2002) supplemented to also contain 10%fetal calf serum (FCS). Cells were incubated at 37° C. in an atmospherewith 5% CO₂ in a humidified incubator. For transfection of HepG2.2.15cells with HBV targeting siRNAs, cells were seeded at a density of 15000cells/well in 96-well regular tissue culture plates. Transfection ofcells was carried out using RNAiMAX (Invitrogen/Life Technologies)according to the manufacturer's instructions. Dose-response experimentswere done with oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625,0.3125, 0.15625 and 0.07813 nM. For each HBV targeting siRNA treatment(e.g., ds-siRNA, as identified by the ds-siNA ID in Table 6), four wellswere transfected in parallel, and individual data points were collectedfrom each well. After 24 h of incubation with siRNA, media was removed,and cells were lysed and analyzed with a QuantiGene2.0 branched DNA(bDNA) probe set specific for HBV genotype D (also called Hepatitis Bvirus subtype ayw, complete genome of 3182 base-pairs) as present incell line HepG2.2.15.

For each well, the HBV on-target mRNA levels were normalized to theGAPDH mRNA level. As shown in Table 6, the activity of the HBV targetingds-siRNAs was expressed as EC50, 50% reduction of normalized HBV RNAlevel from no drug control. As shown in Table 6, the cytotoxicity of theHBV targeting ds-siRNAs was expressed by CC50 of 50% reduction of GAPDHmRNA from no drug control.

Example 3. Use of Ds-siNAs to Treat Hepatitis B Virus Infection

In this example, the ds-siNAs synthesized in Example 1 are used to treata hepatitis B virus infection in a subject. Generally, a compositioncomprising a ds-siNA from Table 6 (as identified by the ds-siNA ID) anda pharmaceutically acceptable carrier is administered to the subjectsuffering from hepatitis B virus. The ds-siNA from Table 6 is conjugatedto N-acetylgalactosamine. The ds-siNA is administered at a dose of 0.3to 5 mg/kg every three weeks by subcutaneous injection or intravenousinfusion.

Example 4. Ds-siNA Hepatitis B Clinical Trial

In this example, the ds-siNAs from Tables 6A and 6B (as identified bythe ds-siNA ID) will be evaluated for safety and efficacy in healthyvolunteers and chronic hepatitis B patients.

ds-siNAs are being developed for the treatment of chronic hepatitis B(CHB) in adults. The study will be conducted in 3 parts, a singleascending-dose (SAD) phase in healthy volunteers (Group A), asingle-dose (SD) phase in patients with CHB (Group B), and a multipleascending-dose (MAD) phase in patients with CHB (Group C).

Study Design

Study Type: Interventional (Clinical Trial) Estimated 50 participantsEnrollment: Allocation: Randomized Intervention Sequential AssignmentModel: Intervention Progression from the SAD phase to the first cohortModel in the MAD phase is contingent upon the Safety Description: ReviewCommittee (SRC) review of a minimum of 14 days post-dose safety andtolerability data from all HV in at least the first 2 SAD cohorts. TheSRC will select one (or more) well-tolerated dose(s) from the SAD phasefor administration in the SD and MAD phases. In all study phases, dosingwill be staggered with the use of sentinel participants to allow timefor the assessment of safety before additional subjects are exposed tostudy drug. Masking: Triple (Participant, Care Provider, Investigator)Masking This is a double-blind placebo-controlled study in Description:which the study site team, the Sponsor, and the participants will beblinded to treatment assignment. The unblinded pharmacist will covereach syringe, prior to transport to the bedside, to ensure blinding.Participants will be centrally assigned to randomized study interventionusing an Interactive Voice/Web Response System (IVRS/IWRS). PrimaryTreatment Purpose:

Arm Intervention/treatment Experimental: Cohort A1 ds-siNA Drug: ds-siNASingle dose, Subcutaneous injection of ds-siNA is a syntheticribonucleic acid 0.1 mg/kg of ds-siNA (HV) interference (RNAi) drug thatconsists of double- stranded oligonucleotides conjugated to an N-acetyl-D-galactosamine (GalNAc) ligand. ds- siNA, sterile solution ofthe ds-siNA at a concentration of 185 mg/mL in water for injection(WFI). Placebo Comparator: Cohort A1 Drug: Placebo for ds-siNA PlaceboSterile 9% saline for injection. Single dose, Subcutaneous injection ofOther Name: Placebo 0.1 mg/kg of Placebo for ds-siNA (HV) Experimental:Cohort A2 ds-siNA Drug: ds-siNA Single dose, Subcutaneous injection ofds-siNA is a synthetic ribonucleic acid 1.5 mg/kg of ds-siNA (HV)interference (RNAi) drug that consists of double- strandedoligonucleotides conjugated to an N- acetyl-D-galactosamine (GalNAc)ligand. ds- siNA, sterile solution of the ds-siNA at a concentration of185 mg/mL in water for injection (WFI). Placebo Comparator: Cohort A2Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection.Single dose, Subcutaneous injection of Other Name: Placebo 1.5 mg/kg ofPlacebo for ds-siNA (HV) Experimental: Cohort A3 ds-siNA Drug: ds-siNASingle dose, Subcutaneous injection of ds-siNA is a syntheticribonucleic acid 3 mg/kg of ds-siNA (HV) interference (RNAi) drug thatconsists of double- stranded oligonucleotides conjugated to an N-acetyl-D-galactosamine (GalNAc) ligand. ds- siNA, sterile solution ofthe ds-siNA at a concentration of 185 mg/mL in water for injection(WFI). Placebo Comparator: Cohort A3 Drug: Placebo for ds-siNA PlaceboSterile 9% saline for injection. Single dose, Subcutaneous injection ofOther Name: Placebo 3 mg/kg of Placebo for ds-siNA (HV) Experimental:Cohort A4 ds-siNA Drug: ds-siNA Single dose, Subcutaneous injection ofds-siNA is a synthetic ribonucleic acid 6 mg/kg of ds-siNA (HV)interference (RNAi) drug that consists of double- strandedoligonucleotides conjugated to an N- acetyl-D-galactosamine (GalNAc)ligand. ds- siNA, sterile solution of the ds-siNA at a concentration of185 mg/mL in water for injection (WFI). Placebo Comparator: Cohort A4Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection.Single dose, Subcutaneous injection of Other Name: Placebo 6 mg/kg ofPlacebo for ds-siNA (HV) Experimental: Cohort A5 ds-siNA Drug: ds-siNASingle dose, Subcutaneous injection of ds-siNA is a syntheticribonucleic acid 12 mg/kg of ds-siNA (HV) interference (RNAi) drug thatconsists of double- stranded oligonucleotides conjugated to an N-acetyl-D-galactosamine (GalNAc) ligand. ds- siNA, sterile solution ofthe ds-siNA at a concentration of 185 mg/mL in water for injection(WFI). Placebo Comparator: Cohort A5 Drug: Placebo for ds-siNA PlaceboSterile 9% saline for injection. Single dose, Subcutaneous injection ofOther Name: Placebo 12 mg/kg of Placebo for ds-siNA (HV) Experimental:Cohort B ds-siNA Drug: ds-siNA Single dose, Subcutaneous injection ofds-siNA is a synthetic ribonucleic acid 3 mg/kg of for ds-siNA (NUCnaïve, interference (RNAi) drug that consists of double- CHB) strandedoligonucleotides conjugated to an N- acetyl-D-galactosamine (GalNAc)ligand. ds- siNA, sterile solution of the ds-siNA at a concentration of185 mg/mL in water for injection (WFI). Placebo Comparator: Cohort BDrug: Placebo for ds-siNA Placebo Sterile 9% saline for injection.Single dose, Subcutaneous injection of Other Name: Placebo 3 mg/kg ofPlacebo for ds-siNA (NUC naïve, CHB) Experimental: Cohort C1 ds-siNADrug: ds-siNA 4 doses-Subcutaneous injection of ds-siNA is a syntheticribonucleic acid 1.5 mg/kg of ds-siNA administered interference (RNAi)drug that consists of double- every 28 days (NUC experienced, strandedoligonucleotides conjugated to an N- CHB) acetyl-D-galactosamine(GalNAc) ligand. ds- siNA, sterile solution of the ds-siNA at aconcentration of 185 mg/mL in water for injection (WFI). PlaceboComparator: Cohort C1 Drug: Placebo for ds-siNA Placebo Sterile 9%saline for injection. 4 doses-Subcutaneous injection of Other Name:Placebo 1.5 mg/kg of Placebo for ds-siNA administered every 28 days (NUCexperienced, CHB) Experimental: Cohort C2 ds-siNA Drug: ds-siNA 4doses-Subcutaneous injection of ds-siNA is a synthetic ribonucleic acid3 mg/kg of ds-siNA administered every interference (RNAi) drug thatconsists of double- 28 days (NUC experienced, CHB) strandedoligonucleotides conjugated to an N- acetyl-D-galactosamine (GalNAc)ligand. ds- siNA, sterile solution of the ds-siNA at a concentration of185 mg/mL in water for injection (WFI). Placebo Comparator: Cohort C2Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection. 4doses-Subcutaneous injection of Other Name: Placebo 3 mg/kg of Placebofor ds-siNA administered every 28 days (NUC experienced, CHB)Experimental: Cohort C3 ds-siNA Drug: ds-siNA 4 doses-Subcutaneousinjection of ds-siNA is a synthetic ribonucleic acid 6 mg/kg of ds-siNAadministered every interference (RNAi) drug that consists of double- 28days (NUC experienced, CHB) stranded oligonucleotides conjugated to anN- acetyl-D-galactosamine (GalNAc) ligand. ds- siNA, sterile solution ofthe ds-siNA at a concentration of 185 mg/mL in water for injection(WFI). Placebo Comparator: Cohort C3 Drug: Placebo for ds-siNA PlaceboSterile 9% saline for injection. 4 doses-Subcutaneous injection of OtherName: Placebo 6 mg/kg of Placebo for ds-siNA administered every 28 days(NUC experienced, CHB)

Outcome Measures

Primary Outcome Measures:

Number of healthy volunteers with Adverse Events as assessed by CTCAEv5.0 [Time Frame: 4 weeks]

Number of participants with abnormalities in vital signs,electrocardiogram (ECG), and clinically significant laboratory findings

Number participants with non-cirrhotic chronic Hepatitis B with AdverseEvents as assessed by CTCAE v5.0 [Time Frame: 16 weeks]

Number of participants with abnormalities in vital signs,electrocardiogram (ECG), and clinically significant laboratory findings.

Secondary Outcome Measures:

To characterize the pharmacokinetics of ds-siNA in healthy volunteers bymonitoring plasma pharmacokinetics profiles of [Time Frame: 4 weeks]Measure the amount of ds-siNA excreted in urine

To characterize the pharmacokinetics of ds-siNA in healthy volunteers bymonitoring through concentrations of [Time Frame: 4 weeks]

Measure the amount of ds-siNA renal clearance (CLR).

To characterize the pharmacokinetics of ds-siNA in participants withnon-cirrhotic CHB by monitoring plasma pharmacokinetics profiles ofds-siNA. [Time Frame: 12 weeks]

Measure the amount of ds-siNA excreted in urine

To characterize the pharmacokinetics of ds-siNA in participants withnon-cirrhotic CHB by monitoring through concentrations of ds-siNA. [TimeFrame: 12 weeks]

Measure ds-siNA renal clearance (CLR).

Other Outcome Measures:

To evaluate the preliminary antiviral efficacy of ds-siNA inparticipants with CHB by monitoring changes in serum HBsAg levels (allGroup B and C participants) during and after single dose and 12 weeks oftreatment with DCR HBVS. [Time Frame: 12 weeks]

Proportion of participants achieving at least a 1-log reduction in HBsAgand achieving a HBsAg level <100 IU/mL at last scheduled visit Time toHBsAg loss (Kaplan-Mayer) Time to anti-HBs seroconversion

To evaluate the preliminary antiviral efficacy of ds-siNA inparticipants with CHB by monitoring HBeAg levels (HBeAg+participantsonly) during and after single dose and 12 weeks of treatment with DCRHBVS. [Time Frame: 12 weeks]

% of participants with HBeAg loss and anti HBe at last scheduled visit(if HBeAg positive at study entry)

To evaluate the preliminary antiviral efficacy of ds-siNA inparticipants with CHB by monitoring HBV DNA levels (all Group B and Cparticipants) during and after single dose and 12 weeks of treatmentwith DCR HBVS. [Time Frame: 12 weeks]

Proportion of participants achieving HBV DNA <2000 IU/mL (if >2,000IU/mL at Baseline); and proportion of participants achievingPCR-nondetectable HBV DNA (if HBV DNA was detectable at Baseline).

To characterize the pharmacodynamics (PD) of ds-siNA on plasma levels ofHBsAg and HBV in blood. [Time Frame: 12 weeks]

Track post-treatment duration of any observed efficacy effects.

Eligibility Criteria

Ages Eligible for Study: 18 Years to 65 Years (Adult, Older Adult) SexesEligible for Study: All Accepts Healthy Volunteers: Yes

Inclusion Criteria:

Healthy at the time of screening as determined by medical evaluation.

Capable of giving informed consent.

12-lead ECG within normal limits or with no clinically significantabnormalities.

Negative screen for alcohol or drugs of abuse.

Non-smokers for at least 3 months with a negative urinary cotinineconcentration at screening.

BMI within range 18.0-32.0 kg/m2 (inclusive).

Female participants not pregnant, not breastfeeding, and not ofchildbearing potential or willing to follow contraceptive guidance.

Chronic hepatitis B infection (Group B and C only).

Clinical history compatible with compensated liver disease with noevidence of cirrhosis (Group B and C only).

Continuously on nucleotides (NUC) therapy for at least 12 weeks prior toscreening (Group C only).

Exclusion Criteria:

-   -   History of any medical condition that may interfere with the        absorption, distribution, or elimination of study drug.

Poorly controlled or unstable hypertension.

History of diabetes mellitus treated with insulin or hypoglycemicagents.

History of asthma requiring hospital admission within the preceding 12months.

Evidence of G-6-PD deficiency.

Currently poorly controlled endocrine conditions, excluding thyroidconditions.

History of multiple drug allergies or history of allergic reaction to anoligonucleotide or GalNAc.

Clinically relevant surgical history.

Use of prescription medications (excluding contraception for women)within 4 weeks prior to the administration of study intervention.

Use of clinically relevant over-the-counter medication or supplements(excluding routine vitamins) within 7 days of first dosing.

Has received an investigational agent within the 3 months prior todosing or is in follow-up of another study.

Antiviral therapy (other than entecavir or tenofovir) within 3 months ofscreening or treatment with interferon in the last 3 years (Group B andC only).

Use within the last 6 months of anticoagulants or systemicallyadministered corticosteroids, immunomodulators, or immunosuppressants(Group B and C only).

Example 5: Synthesis of 5′ End Cap Monomer

Example 5 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1(15 g, 57.90 mmol) in DMF (150 mL)were added AcSK (11.24 g, 98.43 mmol) and TBAI (1.07 g, 2.89 mmol), andthe mixture was stirred at 25° C. for 12 h. Upon completion as monitoredby LCMS, the mixture was diluted with H₂O (10 mL) and extracted with EA(200 mL * 3). The combined organic layers were washed with brine (200mL * 3), dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to give 2 (14.5 g, 96.52% yield, 98% purity) as acolorless oil. ESI-LCMS: 254.28 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃)δ=4.78-4.65 (m, 2H), 3.19 (d, J=14.1 Hz, 2H), 2.38 (s, 3H), 1.32 (t,J=6.7 Hz, 12H); ³¹P NMR (162 MHz, CDCl₃) δ=20.59.

Preparation of (3): To a solution of 2 (14.5 g, 57.02 mmol) in CH₃CN (50mL) and MeOH (25 mL) was added NaOH (3 M, 28.51 mL), and the mixture wasstirred at 25° C. for 12 h under Ar. Upon completion as monitored byTLC, the reaction mixture was concentrated under reduced pressure toremove CH₃CN and CH₃₀H. The residue was diluted with water (50 mL) andadjust pH=7 by 6M HCl, and the mixture was extracted with EA (50 mL *3). The combined organic layers were washed with brine (50 mL * 3),dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to give 3 (12.1 g, crude) as a colorless oil.

Preparation of (4): To a solution of 3 (12.1 g, 57.01 mmol) in CH₃CN (25mL) and MeOH (25 mL) was added A (14.77 g, 57.01 mmol) dropwise at 25°C., and the mixture was stirred at 25° C. under Ar for 12 h. Uponcompletion as monitored by LCMS, the reaction mixture was concentratedunder reduced pressure to give 4 (19.5 g, 78.85% yield) as a colorlessoil. ¹H NMR (400 MHz, CDCl₃) δ=4.80-4.66 (m, 4H), 2.93 (d, J=11.3 Hz,4H), 1.31 (dd, J=3.9, 6.1 Hz, 24H); ³¹P NMR (162 MHz, CDCl₃) δ=22.18.

Preparation of (5): To a solution of 4 (19.5 g, 49.95 mmol) in MeOH (100mL) and H₂O (100 mL) was added Oxone (61.41 g, 99.89 mmol) at 25° C. inportions, and the mixture was stirred at 25° C. for 12 h under Ar. Uponcompletion as monitored by LCMS, the reaction mixture was filtered, andthe filtrate was concentrated under reduced pressure to remove MeOH. Theresidue was extracted with EA (50 mL *3). The combined organic layerswere washed with brine (50 mL * 3), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Thecrude product was triturated with i-Pr₂O and n-Hexane (1:2, 100 mL) at25° C. for 30 min to give 5 (15.6 g, 73.94% yield,) as a white solid. ¹HNMR (400 MHz, CDCl₃) δ=4.92-4.76 (m, 4H), 4.09 (d, J=16.1 Hz, 4H), 1.37(dd, J=3.5, 6.3 Hz, 24H); ³¹P NMR (162 MHz, CDCl₃) δ=10.17.

Preparation of (7): To a mixture of 5 (6.84 g, 16.20 mmol) in THF (20mL) was added LiBr (937.67 mg, 10.80 mmol) until dissolved, followed byDIEA (1.40 g, 10.80 mmol, 1.88 mL) under argon at 15° C. The mixture wasstirred at 15° C. for 15 min. 6 (4 g, 10.80 mmol) were added. Themixture was stirred at 15° C. for 3 h. Upon completion as monitored byLCMS, the reaction mixture was quenched by addition of H₂O (40 mL) andextracted with EA (40 mL * 3). The combined organic layers were washedwith brine (100 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. The residue was purified by flashreverse-phase chromatography (120 g C-18 Column, Eluent of 0-60% ACN/H₂Ogradient @ 80 mL/min) to give 7 (5.7 g, 61.95% yield) as a colorlessoil. ESI-LCMS: 611.2 [M+H]⁺; H NMR (400 MHz, CDCl₃); δ=9.26 (s, 1H),7.50 (d, J=8.1 Hz, 1H), 7.01 (s, 2H), 5.95 (d, J=2.7 Hz, 1H), 5.80 (dd,J=2.1, 8.2 Hz, 1H), 4.89-4.72 (m, 2H), 4.66 (d, J=7.2 Hz, 1H), 4.09-4.04(m, 1H), 3.77 (dd, J=2.7, 4.9 Hz, 1H), 3.62 (d, J=3.1 Hz, 1H), 3.58 (d,J=3.1 Hz, 1H), 3.52 (s, 3H), 1.36 (td, J=1.7, 6.1 Hz, 12H), 0.92 (s,9H), 0.12 (s, 6H); ³¹P NMR (162 MHz, CDCl₃) δ=9.02

Preparation of (8): To a mixture of 7 (5.4 g, 8.84 mmol) in THF (80 mL)was added Pd/C (5.4 g, 10% purity) under N₂. The suspension was degassedunder vacuum and purged with H₂ several times. The mixture was stirredunder H₂ (15 psi) at 20° C. for 1 hr. Upon completion as monitored byLCMS, the reaction mixture was filtered, and the filtrate wasconcentrated to give 8 (5.12 g, 94.5% yield) as a white solid. ESI-LCMS:613.3 [M+H]⁺; H NMR (400 MHz, CD3CN) δ=9.31 (s, 1H), 7.37 (d, J=8.0 Hz,1H), 5.80-5.69 (m, 2H), 4.87-4.75 (m, 2H), 4.11-4.00 (m, 1H), 3.93-3.85(m, 1H), 3.80-3.74 (m, 1H), 3.66-3.60 (m, 1H), 3.57-3.52 (m, 1H), 3.49(s, 3H), 3.46-3.38 (m, 1H), 2.35-2.24 (m, 1H), 2.16-2.03 (m, 1H),1.89-1.80 (m, 1H), 1.37-1.34 (m, 12H), 0.90 (s, 9H), 0.09 (s, 6H); ³¹PNMR (162 MHz, CD3CN) δ=9.41.

Preparation of (9): To a solution of 8 (4.4 g, 7.18 mmol) in THF (7.2mL) was added TBAF (1 M, 7.18 mL), and the mixture was stirred at 20° C.for 1 hr. Upon completion as monitored by LCMS, the reaction mixture wasdiluted with H₂O (50 mL) and extracted with EA (50 mL*4). The combinedorganic layers were washed with brine (50 mL), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by flash silica gel chromatography (ISCO®; 40 gSepaFlash@Silica Flash Column, Eluent of 0-5%, MeOH/DCM gradient @ 40mL/min) to give 9 (3.2 g, 88.50% yield) as a white solid. ESI-LCMS:499.2 [M+H]⁺1; ¹H NMR (400 MHz, CD3CN) δ=9.21 (s, 1H), 7.36 (d, J=8.3Hz, 1H), 5.81-5.72 (m, 2H), 4.88-4.74 (m, 2H), 3.99-3.87 (m, 2H), 3.84(dd, J=1.9, 5.4 Hz, 1H), 3.66-3.47 (m, 7H), 2.98 (s, 1H), 2.44-2.15 (m,2H), 1.36 (d, J=6.0 Hz, 12H); ³¹P NMR (162 MHz, CD3CN) δ=9.48.

Preparation of (Example 5 monomer): To a mixture of 9 (3.4 g, 6.82 mmol,1 eq) and 4A MS (3.4 g) in MeCN (50 mL) was added P1(2.67 g, 8.87 mmol,2.82 mL, 1.3 eq) at 0° C., followed by addition of1H-imidazole-4,5-dicarbonitrile (886.05 mg, 7.50 mmol) at 0° C. Themixture was stirred at 20° C. for 2 h. Upon completion as monitored byLCMS, the reaction mixture was quenched by addition of saturated aq.NaHCO₃(50 mL) and diluted with DCM (100 mL). The organic layer waswashed with saturated aq. NaHCO₃(50 mL * 2), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give a residue. The residuewas purified by prep-HPLC: column: YMC-Triart Prep C18 250*50 mm*10 um;mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B %: 15% to give a impureproduct. The impure product was further purified by a flash silica gelcolumn (0% to 5% i-PrOH in DCM with 0.5% TEA) to give Example 5 monomer(2.1 g, 43.18% yield) as a white solid. ESI-LCMS: 721.2 [M+Na]⁺; H NMR(400 MHz, CD3CN) δ=9.29 (s, 1H), 7.45 (d, J=8.1 Hz, 1H), 5.81 (d, J=4.2Hz, 1H), 5.65 (d, J=8.1 Hz, 1H), 4.79-4.67 (m, 2H), 4.26-4.05 (m, 2H),4.00-3.94 (m, 1H), 3.89-3.63 (m, 6H), 3.53-3.33 (m, 5H), 2.77-2.61 (m,2H), 2.31-2.21 (m, 1H), 2.16-2.07 (m, 1H), 1.33-1.28 (m, 12H), 1.22-1.16(m, 1H), 1.22-1.16 (m, 11H); ³¹P NMR (162 MHz, CD3CN) δ=149.89, 149.78,10.07, 10.02.

Example 6. Synthesis of 5′ End Cap Monomer

Example 6 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1(5 g, 13.42 mmol) in DMF (50 mL)were added PPh₃ (4.58 g, 17.45 mmol) and 2-hydroxyisoindoline-1,3-dione(2.85 g, 17.45 mmol), followed by a solution of DIAD (4.07 g, 20. 13mmol, 3.91 mL) in DMF (10 mL) dropwise at 15° C. The resulting solutionwas stirred at 15° C. for 18 hr. The reaction mixture was then dilutedwith DCM (50 mL), washed with H₂O (60 mL*3) and brine (30 mL), driedover Na₂SO₄, filtered and evaporated to give a residue. The residue wasthen triturated with EtOH (55 mL) for 30 min, and the collected whitepowder was washed with EtOH (10 mL*2) and dried to give 2 (12.2 g, 85.16% yield) as a white powder (the reaction was set up in two batches andcombined) ESI-LCMS: 518.1 [M+H]⁺.

Preparation of (3): 2 (6 g, 11.59 mmol) was suspended in MeOH (50 mL),and then NH₂NH₂.H₂O (3.48 g, 34. 74 mmol, 3.38 mL, 50% purity) was addeddropwise at 20° C. The reaction mixture was stirred at 20° C. for 4 hr.Upon completion, the reaction mixture was diluted with EA (20 mL) andwashed with NaHCO₃(10 mL*2) and brine (10 mL). The combined organiclayers were then dried over Na₂SO₄, filtered and evaporated to give 3(8.3 g, 92.5% yield) as a white powder. (The reaction was set up in twobatches and combined). ESI-LCMS: 388.0 [M+H]⁺; H NMR (400 MHz, DMSO-d6)δ=11.39 (br s, 1H), 7.72 (d, J=8.1 Hz, 1H), 6.24-6.09 (m, 2H), 5.80 (d,J=4.9 Hz, 1H), 5.67 (d, J=8.1 Hz, 1H), 4.26 (t, J=4.9 Hz, 1H), 4.03-3.89(m, 1H), 3.87-3.66 (m, 3H), 3.33 (s, 3H), 0.88 (s, 9H), 0.09 (d, J=1.3Hz, 6H)

Preparation of (4): To a solution of 3 (7 g, 18.06 mmol) and Py (1.43 g,18.06 mmol, 1.46 mL) in DCM (130 mL) was added a solution of MsCl (2.48g, 21.68 mmol, 1. 68 mL) in DCM (50 mL) dropwise at −78° C. under N₂.The reaction mixture was allowed to warm to 15° C. in 30 min and stirredat 15° C. for 3 h. The reaction mixture was quenched by addition ofice-water (70 mL) at 0° C., and then extracted with DCM (50 mL * 3). Thecombined organic layers were washed with saturated aq. NaHCO₃(50 mL) andbrine (30 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. The residue was purified by flashsilica gel chromatography (ISCO®; 30 g SepaFlash@Silica Flash Column,Eluent of 0-20% i-PrOH/DCM gradient @ 30 mL/min to give 4 (6.9 g, 77.94%yield) as a white solid. ESI-LCMS: 466.1 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d) δ=11.41 (br s, 1H), 10. 15 (s, 1H), 7. 69 (d, J=8.1 Hz, 1H),5.80 (d, J=4.4 Hz, 1H), 5.65 (d, J=8. 1 Hz, 1H), 4.24 (t, J=5.2 Hz, 1H),4.16-3.98 (m, 3H), 3. 87 (t, J=4.8 Hz, 1H), 3.00 (s, 3H), 2.07 (s, 3H),0.88 (s, 9H), 0. 10 (d, J=1.5 Hz, 6H)

Preparation of (5): To a solution of 4 (6.9 g, 14.82 mmol) in THF (70mL) was added TBAF (1 M, 16.30 mL) at 15° C. The reaction mixture wasstirred at 15° C. for 18 hr, and then evaporated to give a residue. Theresidue was purified by flash silica gel chromatography (ISCO@; 24 gSepaFlash@ Silica Flash Column, Eluent of 0˜9% MeOH/Ethyl acetategradient @ 30 mL/min) to give 5 (1.8 g, 50.8% yield) as a white solid.ESI-LCMS: 352.0 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ=11.40 (s, 1H), 10.13(s, 1H), 7.66 (d, J=8.1 Hz, 1H), 5.83 (d, J=4. 9 Hz, 1H), 5.65 (dd, J=1.8, 8. 1 Hz, 1H), 5.36 (d, J=6. 2 Hz, 1H), 4.13-4.00 (m, 4H), 3. 82 (t,J=5.1 Hz, 1H), 3.36 (s, 3H), 3.00 (s, 3H)

Preparation of (Example 6 monomer): To a mixture of 5 (3 g, 8.54 mmol)and DIEA (2.21 g, 17.08 mmol, 2.97 mL) in ACN (90 mL) was added P2 (3.03g, 12.81 mmol) dropwise at 15° C. The reaction mixture was stirred at15° C. for 5 h. Upon completion, the reaction mixture was diluted withEA (40 mL) and quenched with 5% NaHCO₃(20 mL). The organic layer waswashed with brine (30 mL), dried over Na₂SO₄, filtered and evaporated togive a residue. The residue was purified by flash silica gelchromatography (ISCO®; 12 g SepaFlash@ Silica Flash Column, Eluent of0-15% i-PrOH/(DCM with 2% TEA) gradient @ 20 mL/min) to Example 6monomer (2.1 g, 43.93% yield) as a white solid. ESI-LCMS: 552.3 [M+H]⁺;¹H NMR (400 MHz, CD3CN) δ=8.78 (br s, 1H), 7.57 (dd, J=4.6, 8.2 Hz, 1H),5.97-5.80 (m, 1H), 5.67 (d, J=8. 3 Hz, 1H), 4.46-4.11 (m, 4H), 3.95-3.58(m, 5H), 3.44 (d, J=16. 3 Hz, 3H), 3.02 (d, J=7. 5 Hz, 3H), 2. 73-2.59(m, 2H), 1.23-1.15 (m, 12H); ³¹P NMR (162 MHz, CD3CN) δ=150.30, 150.10

Example 7: Synthesis of 5′ End Cap Monomer

Example 7 Monomer Synthesis Scheme

Preparation of (2): To the solution of 1 (5 g, 12.90 mmol) and TEA (1.57g, 15.48 mmol, 2.16 mL) in DCM (50 mL) was added P-4 (2.24 g, 15.48mmol, 1.67 mL) in DCM (10 mL) dropwise at 15° C. under N₂. The reactionmixture was stirred at 15° C. for 3 h. Upon completion as monitored byLCMS and TLC (PE: EtOAc=0:1), the reaction mixture was concentrated todryness, diluted with H₂O (20 mL), and extracted with EA (50 mL*3). Thecombined organic layers were washed with brine (30 mL*3), dried overanhydrous Na₂SO₄, filtered, and the filtrate was concentrated underreduced pressure to give a residue. The residue was purified by flashsilica gel chromatography (ISCO®; 40 g SepaFlash@ Silica Flash Column,Eluent of 0-95% Ethyl acetate/Petroleum ether gradient @ 60 mL/min) togive 2 (5.3 g, 71.3% yield) as a white solid. ESI-LCMS: 496.1 [M+H]⁺; HNMR (400 MHz, CDCl₃) δ=0.10 (d, J=4.02 Hz, 6H) 0.91 (s, 9H) 3.42-3.54(m, 3H) 3.65-3.70 (m, 1H) 3.76-3.89 (m, 6H) 4.00 (dd, J=10.92, 2.89 Hz,1H) 4.08-4.13 (m, 1H) 4.15-4.23 (m, 2H) 5.73 (dd, J=8.28, 2.01 Hz, 1H)5.84 (d, J=2.76 Hz, 1H) 6.86 (d, J=15.81 Hz, 1H) 7.72 (d, J=8.03 Hz, 1H)9.10 (s, 1H); ³¹P NMR (162 MHz, CD3CN) 6=9.65

Preparation of (3): To a solution of 2 (8.3 g, 16.75 mmol) in THF (50mL) were added TBAF (1 M, 16.75 mL) and CH₃COOH (1.01 g, 16.75 mmol,957.95 uL). The mixture was stirred at 20° C. for 12 hr. Upon completionas monitored by LCMS, the reaction mixture was concentrated underreduced pressure. The residue was purified by column chromatography(SiO₂, PE: EA=0˜100%; MeOH/EA=0˜10%) to give 3 (5 g, 77.51% yield) as awhite solid. ESI-LCMS: 382.1 [M+H]⁺; H NMR (400 MHz, CDCl₃) δ=3.35 (s,3H) 3.65 (br d, J=2.76 Hz, 3H) 3.68 (d, J=2.76 Hz, 3H) 3.77 (t, J=5.08Hz, 1H) 3.84-4.10 (m, 4H) 5.33 (br d, J=5.52 Hz, 1H) 5.62 (d, J=7.77 Hz,1H) 5.83 (d, J=4.94 Hz, 1H) 7.69 (d, J=7.71 Hz, 1H) 9.08 (d, J=16.81 Hz,1H) 11.39 (br s, 1H); ³¹P NMR (162 MHz, CD3CN) 6=15.41

Preparation of (Example 7 monomer): To a solution of 3 (2 g, 5.25 mmol)and DIPEA (2.03 g, 15.74 mmol, 2.74 mL, 3 eq) in MeCN (21 mL) andpyridine (7 mL) was added P2 (1.86 g, 7.87 mmol) dropwise at 20° C., andthe mixture was stirred at 20° C. for 3 hr. Upon completion as monitoredby LCMS, the reaction mixture was diluted with water (20 mL) andextracted with EA (50 mL). The combined organic layers were washed withbrine (30 mL), dried over anhydrous Na₂SO₄, filtered, and the filtratewas concentrated under reduced pressure to give a residue. The residuewas purified by flash silica gel chromatography (ISCO®; 25 g SepaFlash@Silica Flash Column, Eluent of 0-45% (Ethyl acetate: EtOH=4:1)/Petroleumether gradient) to give Example 7 monomer (1.2 g, 38.2% yield) as awhite solid. ESI-LCMS: 604.1 [M+H]⁺; ¹H NMR (400 MHz, CD3CN) & 1.12-1.24(m, 12H) 2.61-2.77 (m, 2H) 3.43 (d, J=17.64 Hz, 3H) 3.59-3.69 (m, 2H)3.71-3.78 (m, 6H) 3.79-4.14 (m, 5H) 4.16-4.28 (m, 1H) 4.29-4.42 (m, 1H)5.59-5.72 (m, 1H) 5.89 (t, J=4.53 Hz, 1H) 7.48 (br d, J=12.76 Hz, 1H)7.62-7.74 (m, 1H) 9.26 (br s, 1 H); ³¹P NMR (162 MHz, CD3CN) δ=150.57,149.96, 9.87

Example 8: Synthesis of 5′ End Cap Monomer

Example 8 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1 (30 g, 101.07 mmol, 87% purity)in CH₃CN (1.2 L) and Py (60 mL) were added 12 (33.35 g, 131.40 mmol,26.47 mL) and PPh₃ (37.11 g, 141.50 mmol) in one portion at 10′° C. Thereaction was stirred at 25° C. for 48 h. Upon completion, the mixturewas diluted with saturated aq.Na₂S₂O₃ (300 mL) and saturatedaq.NaHCO₃(300 mL), concentrated to remove CH₃CN, and extracted withEtOAc (300 mL * 3). The combined organic layers were washed with brine(300 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by flash silica gelchromatography (ISCO®; 330 g SepaFlash@ Silica Flash Column, Eluent of0.60%/Methanol/Dichloromethane gradient @ 100 mL/min) to give 2 (28.2 g,72% yield) as a brown solid. ESI-LCMS: 369.1 [M+H]⁺; H NMR (400 MHz,DMSO-d6) δ=11.43 (s, 1H), 7.68 (d, J=8.1 Hz, 1H), 5.86 (d, J=5.5 Hz,1H), 5.69 (d, J=8.1 Hz, 1H), 5.46 (d, J=6.0 Hz, 1H), 4.08-3.96 (m, 2H),3.90-3.81 (m, 1H), 3.60-3.51 (m, 1H), 3.40 (dd, J=6.9, 10.6 Hz, 1H),3.34 (s, 3H).

Preparation of (3): To the solution of 2 (12 g, 32.6 mmol) in DCM (150mL) were added AgNO₃ (11.07 g, 65.20 mmol), 2,4,6-trimethylpyridine(11.85 g, 97.79 mmol, 12.92 mL), and DMTC1 (22.09 g, 65.20 mmol) at 10°C., and the reaction mixture was stirred at 10° C. for 16 hr. Uponcompletion, the mixture was filtered and the filtrate was concentratedunder reduced pressure. The residue was purified by flash silica gelchromatography (ISCO®; 120 g SepaFlash@ Silica Flash Column, Eluent of0˜50% Ethyl acetate/Petroleum ethergradient @ 60 mL/min) to give 3 (17g, 70.78% yield) as a yellow solid. ESI-LCMS: 693.1 [M+Na]⁺¹; H NMR (400MHz, DMSO-d6) δ=11.46 (s, 1H), 7.60 (d, J=8.4 Hz, 1H), 7.49 (d, J=7.2Hz, 2H), 7.40-7.30 (m, 6H), 7.29-7.23 (m, 1H), 6.93 (d, J=8.8 Hz, 4H),5.97 (d, J=6.0 Hz, 1H), 5.69 (d, J=8.0 Hz, 1H), 4.05-4.02 (m, 1H), 3.75(d, J=1.2 Hz, 6H), 3.57 (t, J=5.6 Hz, 1H), 3.27 (s, 4H), 3.06 (t, J=10.4Hz, 1H), 2.98-2.89 (m, 1H).

Preparation of (4): To a solution of 3 (17 g, 25.35 mmol) in DMF (200mL) was added AcSK (11.58 g, 101.42 mmol) at 25° C., and the reactionwas stirred at 60° C. for 2 hr. The mixture was diluted with H₂O (600mL) and extracted with EtOAc (300 mL * 4). The combined organic layerswere washed with brine (300 mL), dried over Na₂SO₄, filtered, andconcentrated under reduced pressure to give 4 (15.6 g, crude) as a brownsolid, which was used directly without further purification. ESI-LCMS:641.3 [M+H]⁺.

Preparation of (5): To a solution of 4 (15.6 g, 25.21 mmol) in CH₃CN(200 mL) were added DTT (11.67 g, 75.64 mmol, 11.22 mL) and LiOH.H₂O(1.06 g, 25.21 mmol) at 10° C. under Ar. The reaction was stirred at 10°C. for 1 hr. The mixture was concentrated under reduced pressure toremove CH₃CN, and the residue was diluted with H₂O (400 mL) andextracted with EtOAc (200 mL * 3). The combined organic layers werewashed with brine (300 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was purified byflash silica gel chromatography (ISCO®; 220 g SepaFlash@ Silica FlashColumn, Eluent of 0˜60% Ethyl acetate/Petroleum ether gradient @ 100mL/min) to give 5 (8.6 g, 56.78% yield) as a white solid. ESI-LCMS:599.3 [M+Na]⁺; ¹H NMR (400 MHz, DMSO-d6) δ=8.79 (s, 1H), 7.61 (d, .,8.0Hz, 1H), 7.56-7.46 (m, 2H), 7.45-7.37 (m, 4H), 7.36-7.27 (m, 3H), 6.85(dd, J=2.8, 8.8 Hz, 4H), 5.85 (d, J=1.3 Hz, 1H), 5.68 (dd, j=2.0, 8.2Hz, 1H), 4.33-4.29 (m, 1H), 3.91 (dd, J=4.8, 8.2 Hz, 1H), 3.81 (d, J=1.6Hz, 6H), 3.33 (s, 3H), 2.85-2.80 (m, 1H), 2.67-2.55 (m, 2H), 1.11(t,.J=8.8 Hz, 1H).

Preparation of (Example 8 monomer): To a solution of 5 (6 g, 10.40 mmol)in DCM (120 mL) were added P1(4.08 g, 13.53 mmol, 4.30 mL) and DCI (1.35g, 11.45 mmol) in one portion at 10° C. under Ar. The reaction wasstirred at 10° C. for 2 hr. The reaction mixture was diluted withsaturated aq.NaHCO₃(50 mL) and extracted with DCM (20 mL * 3). Thecombined organic layers were washed with brine (30 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (column: YMC-Triart PrepC18 250*50 mm*10 um; mobile phase: [water(10 mM NH₄HCO₃)-ACN]; B %:35%-81%,20 min) to give Example 8 monomer (3.54 g, 43.36% yield) as ayellow solid. ESI-LCMS: 776.4 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6)δ=7.65-7.38 (m, 7H), 7.37-7.22 (m, 3H), 6.90 (d, J=8.4 Hz, 4H), 5.92 (s,1H), 5.66 (t, J=8.2 Hz, 1H), 4.13 (d, J=4.0 Hz, 1H), 4.00-3.88 (m, 1H),3.87-3.59 (m, 10H), 3.33 (d, J=5.8 Hz, 3H), 3.12-2.94 (m, 1H), 2.78-2.60(m, 3H), 2.55-2.48 (m, 1H), 1.36-0.98 (m, 12H); ³¹P NMR (162 MHz,DMSO-d6) δ=162.69.

Example 9: Synthesis of 5′ End Cap Monomer

Example 9 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1 (22.6 g, 45.23 mmol) in DCM (500mL) and H₂O (125 mL) were added TEMPO (6.40 g, 40.71 mmol) and DIB(29.14 g, 90.47 mmol) at 0° C. The mixture was stirred at 20° C. for 20h. Upon completion as monitored by LCMS, saturated aq. NaHCO₃ was addedto the mixture to adjust pH>8. The mixture was diluted with H₂O (200 mL)and washed with DCM (100 mL * 3). The aqueous layer was collected,adjusted to pH<5 by HCl (4M), and extracted with DCM (200 mL * 3). Thecombined organic layers were washed with brine (300 mL), dried overNa₂SO₄, filtered, and concentrated under reduced pressure to give 2(17.5 g, 68.55% yield) as a yellow solid. ESI-LCMS: 514.2 [M+H]⁺; ¹H NMR(400 MHz, DMSO-d6) δ=11.27 (s, 1H), 8.86 (s, 1H), 8.78 (s, 1H), 8.06 (d,J=7.5 Hz, 2H), 7.68-7.62 (m, 1H), 7.59-7.52 (m, 2H), 6.28 (d, J=6.8 Hz,1H), 4.82-4.76 (m, 1H), 4.54 (dd, J=4.1, 6.7 Hz, 1H), 4.48 (d, J=1.8 Hz,1H), 3.32 (s, 3H), 0.94 (s, 9H), 0.18 (d, J=4.8 Hz, 6H).

Preparation of (3): To a solution of 2 (9.3 g, 18.11 mmol) in MeOH (20mL) was added SOCl₂ (3.23 g, 27.16 mmol, 1.97 mL) dropwise at 0° C. Themixture was stirred at 20° C. for 0.5 hr. Upon completion as monitoredby LCMS, the reaction mixture was quenched by addition of saturated aq.NaHCO₃(80 mL) and concentrated under reduced pressure to remove MeOH.The aqueous layer was extracted with DCM (80 mL * 3). The combinedorganic layers were washed with brine (200 mL), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by flash silica gel chromatography (ISCO®; 120 gSepaFlash@ Silica Flash Column, Eluent of 0-5%, MeOH/DCM gradient @ 85mL/min) to give 3 (5.8 g, 60% yield) as a yellow solid. ESI-LCMS: 528.3[M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ=11.28 (s, 1H), 8.79 (d, J=7.3 Hz,2H), 8.06 (d, J=7.5 Hz, 2H), 7.68-7.62 (m, 1H), 7.60-7.53 (m, 2H), 6.28(d, J=6.6 Hz, 1H), 4.87 (dd, J=2.4, 4.0 Hz, 1H), 4.61 (dd, J=4.3, 6.5Hz, 1H), 4.57 (d, J=2.2 Hz, 1H), 3.75 (s, 3H), 3.32 (s, 3H), 0.94 (s,9H), 0.17 (d, J=2.2 Hz, 6H).

Preparation of (4): To a mixture of 3 (5.7 g, 10.80 mmol) in CD30D (120mL) was added NaBD₄ (1.63 g, 43.21 mmol) in portions at 0° C., and themixture was stirred at 20° C. for 1 hr. Upon completion as monitored byLCMS, the reaction mixture was neutralized by AcOH (−10 mL) andconcentrated under reduced pressure to give a residue. The residue waspurified by flash silica gel chromatography (ISCO®; 40 gSepaFlash@Silica Flash Column, Eluent of 0-5%, MeOH/DCM gradient @ 40mL/min) to give 4 (4.15 g, 7.61 mmol, 70.45% yield) as a yellow solid.ESI-LCMS: 502.2 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ=11.23 (s, 1H), 8.76(s, 2H), 8.04 (d, J=7.3 Hz, 2H), 7.69-7.62 (m, 1H), 7.60-7.52 (m, 2H),6.14 (d, J=6.0 Hz, 1H), 5.18 (s, 1H), 4.60-4.51 (m, 2H), 3.98 (d, J=3.0Hz, 1H), 3.32 (s, 3H), 0.92 (s, 9H), 0.13 (d, J=1.5 Hz, 6H).

Preparation of (5): To a solution of 4 (4.85 g, 9.67 mmol) in pyridine(50 mL) was added DMTrCl (5.90 g, 17.40 mmol) at 25° C. and the mixturewas stirred for 2 hr. Upon completion as monitored by LCMS, the reactionmixture was concentrated under reduced pressure to remove pyridine. Theresidue was diluted with EtOAc (150 mL) and washed with H₂O (50 mL * 3),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by flash silica gelchromatography (ISCO®; 80 g SepaFlash@ Silica Flash Column, Eluent of0˜70%, EA/PE gradient @ 60 mL/min) to give 5 (6.6 g, 84.06% yield) as ayellow solid. ESI-LCMS: 804.3[M+H]⁺, ¹H NMR (400 MHz, DMSO-d&) δ=11.22(s, 1H), 8.68 (d, J=11.0 Hz, 2H), 8.03 (d, J=7.3 Hz, 2H), 7.68-7.60 (m,1H), 7.58-7.49 (m, 2H), 7.37-7.30 (m, 2H), 7.27-7.16 (m, 7H), 6.88-6.79(m, 4H), 6.17 (d, J=4.2 Hz, 1H), 4.72 (t, J=5.0 Hz, 1H), 4.60 (t, J=4.5Hz, 1H), 4.03-3.98 (m, 1H), 3.71 (s, 6H), 0.83 (s, 9H), 0.12-0.03 (m,6H).

Preparation of (6): To a solution of 5 (6.6 g, 8.21 mmol) in THF (16 mL)was added TBAF (1 M, 8.21 mL,), and the mixture was stirred at 20° C.for 2 hr. Upon completion as monitored by LCMS, the reaction mixture wasdiluted with EA (150 mL) and washed with H₂O (50 mL*3). The organiclayer was washed with brine (150 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by flash silica gel chromatography (ISCO®; 80 g SepaFlash@Silica Flash Column, Eluent of 10-100%, EA/PE gradient @ 30 mL/min) togive 6 (5.4 g, 94.4% yield) as a yellow solid. ESI-LCMS: 690.3 [M+H]⁺; HNMR (400 MHz, DMSO-d6) δ=11.24 (s, 1H), 8.69 (s, 1H), 8.62 (s, 1H), 8.05(d, J=7.3 Hz, 2H), 7.69-7.62 (m, 1H), 7.60-7.52 (m, 2H), 7.40-7.33 (m,2H), 7.30-7.18 (m, 7H), 6.84 (dd, J=5.9, 8.9 Hz, 4H), 6.19 (d, J=4.8 Hz,1H), 5.36 (d, J=6.0 Hz, 1H), 4.59-4.52 (m, 1H), 4.48 (q, J=5.1 Hz, 1H),4.11 (d, J=4.8 Hz, 1H), 3.72 (d, J=1.0 Hz, 6H), 3.40 (s, 3H).

Preparation of (Example 9 monomer): To a solution of 6 (8.0 g, 11.60mmol) in MeCN (150 mL) was added P-1 (4.54 g, 15.08 mmol, 4.79 mL) at 0°C., followed by DCI (1.51 g, 12.76 mmol) in one portion. The mixture waswarmed to 20° C. and stirred for 2 h. Upon completion as monitored byLCMS, the reaction mixture was quenched by addition of saturated aq.NaHCO₃(50 mL) and diluted with DCM (250 mL). The organic layer waswashed with saturated aq.NaHCO₃(50 mL * 2), dried over Na₂SO₄, filteredand concentrated under reduced pressure. The residue was purified by aflash silica gel column (0% to 60% EA in PE contain 0.5% TEA) to giveExample 9 monomer (5.75 g, 55.37% yield, 99.4% purity) as a white solid.ESI-LCMS: 890.4 [M+H]⁺; ¹H NMR (400 MHz, CD3CN) δ=9.55 (s, 1H),8.63-8.51 (m, 1H), 8.34-8.24 (m, 1H), 7.98 (br d, J=7.5 Hz, 2H),7.65-7.55 (m, 1H), 7.53-7.46 (m, 2H), 7.44-7.37 (m, 2H), 7.32-7.17 (m,7H), 6.84-6.77 (m, 4H), 6.14 (d, J=4.3 Hz, 1H), 4.84-4.73 (m, 1H),4.72-4.65 (m, 1H), 4.34-4.27 (m, 1H), 3.91-3.61 (m, 9H), 3.50-3.43 (m,3H), 2.72-2.61 (m, 1H), 2.50 (t, J=6.0 Hz, 1H), 1.21-1.15 (m, 10H), 1.09(d, J=6.8 Hz, 2H); ³¹P NMR (162 MHz, CD3CN) δ=150.01, 149.65

Example 10: Synthesis of 5′ End Cap Monomer

Example 10 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1(10 g, 27.22 minol) in CH₃CN (200mL) and 1120 (50 mL) were added TEMPO (3.85 g, 24.50 minol) and DIB(17.54 g, 54.44 minol). The mixture was stirred at 25° C. for 12 h. Uponcompletion as monitored by LCMS, the reaction mixture was concentratedunder reduced pressure to give a residue. The residue was trituratedwith EtOAc (600 mL) for 30 min. The resulting suspension was filteredand the collected solid was washed with EtOAc (300 mL*2) to give 2(20.09 g, 91.5% yield) as a white solid. ESI-LCMS: 382.0 [M+H]⁺.

Preparation of (3): To a solution of 2 (6 g, 15.73 mmol) in MeOH (100mL) was added SOCl₂ (2.81 g, 23.60 mmol, 1.71 mL) dropwise at 0° C. Themixture was stirred at 25° C. for 12 h. Upon completion as monitored byLCMS, the reaction mixture was quenched by addition of NaHCO₃(4 g) andstirred at 25° C. for 30 min. The reaction mixture was filtered and thefiltrate was concentrated under reduced pressure to give 3 (18.8 g,95.6% yield) as a white solid. The crude product was used for the nextstep without further purification. (The reaction was set up in parallel3 batches and combined). ESI-LCMS: 396.1 [M+H]⁺;¹H NMR (400 MHz,DMSO-d6) δ=12.26-11.57 (m, 2H), 8.42-8.06 (m, 1H), 6.14-5.68 (m, 2H),4.56 (s, 2H), 4.33 (dd, J=4.0, 7.3 Hz, 1H), 3.77 (m, 3H),3.30 (s, 3H),2.81-2.69 (m, 1H), 1.11 (s, 6H)

Preparation of (4 & 5): To a mixture of 3 (10.1 g, 25.55 mmol) in CD30D(120 mL) was added NaBD₄ (3.29 g, 86.86 mmol, 3.4 eq) in portions at 0°C. The mixture was stirred at 25° C. for 1 h. Upon completion asmonitored by LCMS, the reaction mixture was neutralized with AcOH (˜15mL) and concentrated under reduced pressure to give a residue. Theresidue was purified by flash silica gel chromatography (ISCO®; 120 gSepaFlash@ Silica Flash Column, Eluent of 0-7.4%, MeOH/DCM gradient @ 80mL/min) to give 4 (2.98 g, 6.88 mmol, 27% yield) as a yellow solid.ESI-LCMS: 370.1[M+H]⁺ and 5 (10.9 g, crude) as a yellow solid. ESI-LCMS:300.1[M+H]⁺; ¹H NMR (400 MHz, CD30D) δ=7.85 (s, 1H), 5.87 (d, J=6.0 Hz,1H), 4.46-4.39 (m, 1H), 4.34 (t, J=5.4 Hz, 1H), 4.08 (d, J=3.1 Hz, 1H),3.49-3.38 (m, 4H)

Preparation of 6: To a solution of 4 (1.9 g, 4.58 mmol, 85.7% purity) inpyridine (19 mL) was added DMTrCl (2.02 g, 5.96 mmol). The mixture wasstirred at 25° C. for 2 h under N₂. Upon completion as monitored byLCMS, the reaction mixture was quenched by MeOH (10 mL) and concentratedunder reduce pressure to give a residue. The residue was diluted withH₂O (10 mL*3) and extracted with EA (20 mL*3). The combined organiclayers were washed with brine (20 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduce pressure to give a residue. Theresidue was purified by flash silica gel chromatography (ISCO®; 25 gSepaFlash@ Silica Flash Column, Eluent of 0-77%, PE: (EA with 10% EtOH):1% TEA@ 35 mL/min) to give 6 (2.6 g, 81.71% yield, 96.71% purity) as awhite foam. ESI-LCMS: 672.2 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ=12.02 (s,1H), 7.96 (s, 1H), 7.83 (s, 1H), 7.51 (d, J=7.4 Hz, 2H), 7.37 (d, J=8.6Hz, 4H), 7.25-7.17 (m, 2H), 6.80 (t, J=8.4 Hz, 4H), 5.88 (d, J=6.3 Hz,1H), 4.69 (t, J=5.7 Hz, 1H), 4.64 (s, 1H), 4.54 (s, 1H), 4.19 (d, J=2.9Hz, 1H), 3.77 (d, J=4.5 Hz, 6H), 3.60-3.38 (m, 3H), 2.81 (s, 1H), 1.81(td, J=6.9, 13.7 Hz, 1H), 0.97 (d, J=6.8 Hz, 3H), 0.80 (d, J=6.9 Hz, 3H)

Preparation of Example 10 monomer: To a solution of 6 (8.4 g, 12.5 mmol)in MeCN (80 mL) was added P-1 (4.9 g, 16.26 mmol, 5.16 mL) at 0° C.,followed by addition of DCI (1.624 g, 13.76 mmol) in one portion at 0°C. under Ar. The mixture was stirred at 25° C. for 2 h. Upon completionas monitored by LCMS, the reaction mixture was quenched with saturatedaq.NaHCO₃(20 mL) and extracted with DCM (50 mL*2). The combined organiclayers were dried over anhydrous Na₂SO₄, filtered and concentrated underreduce pressure to give a residue. The residue was purified by flashsilica gel chromatography (ISCO®; 40 g SepaFlash@ Silica Flash Column,Eluent of 0-52% PE: EA (10% EtOH): 5% TEA, @ 80 mL/min) to give Example10 monomer (3.4 g, 72.1% yield,) as a white foam. ESI-LCMS: 872.4[M+H]⁺; ¹H NMR (400 MHz, CD3CN) & 12.46-11.07 (m, 1H), 9.29 (s, 1H),7.84 (d, J=14.6 Hz, 1H), 7.42 (t, J=6.9 Hz, 2H), 7.34-7.17 (m, 7H),6.85-6.77 (m, 4H), 5.95-5.77 (m, 1H), 4.56-4.40 (m, 2H), 4.24 (dd,J=4.0, 13.3 Hz, 1H), 3.72 (d, J=2.0 Hz, 7H), 3.66-3.53 (m, 3H), 3.42 (d,J=11.8 Hz, 3H), 2.69-2.61 (m, 1H), 2.60-2.42 (m, 2H), 1.16-1.00 (m,18H); ³¹P NMR (162 MHz, CD3CN) δ=149.975, 149.9

Example 11: Synthesis of 5′ End Cap Monomer

Example 11 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1 (40 g, 58.16 mmol) in DMF (60 mL)were added imidazole (11.88 g, 174.48 mmol), NaI (13.08 g, 87.24 mmol),and TBSCI (17.52 g, 116.32 mmol) at 20° C. in one portion. The reactionmixture was stirred at 20° C. for 12 h. Upon completion, the mixture wasdiluted with EA (200 mL). The organic layer was washed with brine/water(80 mL/80 mL *4), dried over Na₂SO₄, filtered and evaporated to give 2(50.8 g, crude) as yellow solid. ESI-LCMS: 802.3 [M+H]⁺

Preparation of (3): To a solution of 2 (8.4 g, 10.47 mmol) in DCM (120mL) were added Et₃SiH (3.06 g, 26.3 mmol, 4.2 mL) and TFA (1.29 g, 0.84mL) dropwise at 0° C. The reaction mixture was stirred at 20° C. for 2h. The reaction mixture was washed with saturated aq.NaHCO₃(15 mL) andbrine (80 mL). The organic layer was dried over Na₂SO₄, filtered andevaporated. The residue was purified by flash silica gel chromatography(ISCO@; 80 g SepaFlash@ Silica Flash Column, Eluent of 0˜83% EA/PEgradient @ 80 mL/min) to give 3 (2.92 g, 55.8% yield,) as a white solid.ESI-LCMS: 500.2 [M+H]⁺;^(1I)H NMR (400 MHz, CDCl₃) δ=8.79 (s, 1H), 8.14(s, 1H), 8.02 (d, J=7.6 Hz, 2H), 7.64-7.58 (m, 1H), 7.56-7.49 (m, 2H),5.98-5.93 (m, 1H), 4.63-4.56 (m, 2H), 4.23 (s, 1H), 3.98 (dd, J=1.5,13.1 Hz, 1H), 3.75 (dd, J=1.5, 13.1 Hz, 1H), 3.28 (s, 3H), 2.06-1.99 (m,1H), 1.00-0.90 (m, 9H), 0.15 (d, J=7.0 Hz, 6H).

Preparation of (4): 3 (6 g, 12.01 mmol) and tert-butylN-methylsulfonylcarbamate (3.52 g, 18.01 mmol) were co-evaporated withtoluene (50 mL), dissolved in dry THF (100 mL), and cooled to 0° C. PPh₃(9.45 g, 36.03 mmol,) was then added, followed by dropwise addition ofDIAD (7.28 g, 36.03 mmol, 7.00 mL) in dry THF (30 mL). The reactionmixture was stirred at 20° C. for 18 h. Upon completion, the reactionmixture was then diluted with DCM (100 mL) and washed with water (70 mL)and brine (70 mL), dried over Na₂SO₄, filtered and evaporated to give aresidue. The residue was purified by flash silica gel chromatography(ISCO®; 80 g SepaFlash@ Silica Flash Column, Eluent of 0˜100% Ethylacetate/Petroleum ether gradient @ 60 mL/min) followed by reverse-phaseHPLC (0.1% NH₃.H₂O condition, eluent at 74%) to give 4 (2.88 g, 25%yield) as a white solid. ESI-LCMS: 677.1 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃)δ=9.24 (s, 1H), 8.84 (s, 1H), 8.36 (s, 1H), 8.05 (br d, J=7.3 Hz, 2H),7.66-7.42 (m, 4H), 6.16 (d, J=5.0 Hz, 1H), 4.52 (br t, J=4.5 Hz, 1H),4.25-4.10 (m, 1H), 3.97 (br dd, J=8.0, 14.8 Hz, 1H), 3.48 (s, 3H), 3.27(s, 3H), 1.54 (s, 9H), 0.95 (s, 9H), 0.14 (d, J=0.8 Hz, 6H).

Preparation of (5): To a solution of 4 (2.8 g, 4.14 mmol) in THF (20 mL)was added TBAF (4 M, 1.03 mL) and the mixture was stirred at 20° C. for12 h. The reaction mixture was then evaporated. The residue was purifiedby flash silica gel chromatography (ISCO®; 12 g SepaFlash@ Silica FlashColumn, Eluent of 0-6% MeOH/ethyl acetate gradient @ 20 mL/min) to give5 (2.1 g, 83.92% yield) as a white solid. ESI-LCMS: 563.1[M+H]⁺; ¹H NMR(400 MHz, CDCl₃) δ=8.85-8.77 (m, 1H), 8.38 (s, 1H), 8.11-7.99 (m, 2H),7.64-7.50 (m, 4H), 6.19 (d, J=2.8 Hz, 1H), 4.36-4.33 (m, 1H), 4.29 (brd, J=4.3 Hz, 1H), 4.22-4.02 (m, 2H), 3.65-3.59 (m, 3H), 3.28 (s, 3H),1.54 (s, 9H).

Preparation of (6): To a solution of 5 (2.1 g, 3.73 mmol) in DCM (20 mL)was added TFA (7.70 g, 67.53 mmol, 5 mL) at 0° C. The reaction mixturewas stirred at 20° C. for 24 h. Upon completion, the reaction wasquenched with saturated aq. NaHCO₃ to reach pH 7. The organic layer wasdried over Na₂SO₄, filtered, and evaporated at low pressure. The residuewas purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash@Silica Flash Column, Eluent of 0-7% DCM/MeOH gradient @ 20 mL/min) togive 1.6 g (impure, 75% LCMS purity), followed by prep-HPLC [FAcondition, column: Boston Uni C18 40*150*5 um; mobile phase: [water(0.225% FA)-ACN]; B %: 8%-38%,7.7 min.] to give 6 (1.04 g, 63.7% yield)as a white solid. ESI-LCMS: 485.0 [M+Na]⁺; ¹H NMR (400 MHz, DMSO-d6)δ=11.27-11.21 (m, 1H), 8.77 (s, 1H), 8.74 (s, 1H), 8.05 (d, J=7.3 Hz,2H), 7.68-7.62 (m, 1H), 7.59-7.53 (m, 2H), 7.39 (t, J=6.3 Hz, 1H), 6.16(d, J=6.0 Hz, 1H), 5.48 (d, J=5.5 Hz, 1H), 4.55 (t, J=5.5 Hz, 1H),4.43-4.37 (m, 1H), 4.08-4.02 (m, 1H), 3.41-3.36 (m, 1H), 3.35 (s, 3H),3.31-3.22 (m, 1H), 2.91 (s, 3H).

Preparation of (Example 11 monomer): To a solution of 6 (1 g, 2.16 mmol)in DCM (30 mL) was added P1 (977.58 mg, 3.24 mmol, 1.03 mL), followed byDCI (306.43 mg, 2.59 mmol) at 0° C. in one portion under Ar atmosphere.The mixture was degassed and purged with Ar for 3 times, warmed to 20°C., and stirred for 2 hr under Ar atmosphere. Upon completion asmonitored by LCMS and TLC (PE: EtOAc=4:1), the reaction mixture wasdiluted with sat.aq. NaHCO₃(30 mL) and extracted with DCM (50 mL*2). Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered, andthe filtrate was concentrated under reduced pressure to give a residue.The crude product was purified by reversed-phase HPLC (40 g C18 column:neutral condition, Eluent of 0-57% of 0.3% NH₄HCO₃ in H₂O/CH₃CN ethergradient @ 35 mL/min) to give Example 11 monomer (0.49 g, 33.7% yield)as a white solid. ESI-LCMS: 663.1[M+H]⁺; ¹H NMR (400 MHz, CD3CN)&=1.19-1.29 (m, 12H) 2.71 (q, J=5.77 Hz, 2H) 2.94 (d, J=6.27 Hz, 3H)3.35 (d, J=15.56 Hz, 3H) 3.40-3.52 (m, 2H) 3.61-3.97 (m, 4H) 4.23-4.45(m, 1H) 4.55-4.74 (m, 2H) 6.02 (dd, J=10.67, 6.40 Hz, 1H) 7.25 (br s,1H) 7.47-7.57 (m, 2H) 7.59-7.68 (m, 1H) 8.01 (d, J=7.78 Hz, 2H) 8.28 (s,1H) 8.66 (s, 1H) 9.69 (br s, 1H); ³¹P NMR (162 MHz, CD3CN) δ=150.92,149.78.

Example 12. Synthesis of 5′-Stabilized End Cap Modified Oligonucleotides

This example provides an exemplary method for synthesizing the siNAscomprising a 5′-stabilized end caps disclosed herein. The 5′-stabilizedend cap and/or deuterated phosphoramidites were dissolved in anhydrousacetonitrile and oligonucleotide synthesis was performed on a Expedite8909 Synthesizer using standard phosphoramidite chemistry. An extendedcoupling (12 minutes) of 0.12 M solution of phosphoramidite in anhydrousCH₃CN in the presence of Benzyl-thio-tetrazole (BTT) activator to asolid bound oligonucleotide followed by standard capping, oxidation andsulfurization produced modified oligonucleotides. The 0.02 M 12, THF:Pyridine; Water 7:2:1 was used as an oxidizing agent, while DDTT(dimethylamino-methylidene) amino)-3H-1,2,4-dithiazaoline-3-thione wasused as the sulfur-transfer agent for the synthesis ofoligoribonucleotide with a phosphorothioate backbone. The stepwisecoupling efficiency of all modified phosphoramidites was achieved around98%. After synthesis the solid support was heated with aqueous ammonia(28%) solution at 45° C. for 16 h or 0.05 M K₂CO₃ in methanol was usedto deprotect the base labile protecting groups. The crudeoligonucleotides were precipitated with isopropanol and centrifuged(Eppendorf 5810R, 3000 g, 4° C., 15 min) to obtain a pellet. The crudeproduct was then purified using ion exchange chromatography (TSK gelcolumn, 20 mM NaH₂PO₄, 10% CH₃CN, 1 M NaBr, gradient 20-60% B over 20column volumes) and fractions were analyzed by ion change chromatographyon an HPLC. Pure fractions were pooled and desalted by Sephadex G-25column and evaporated to dryness. The purity and molecular weight weredetermined by HPLC analysis and ESI-MS analysis. Single strand RNAoligonucleotides (sense and antisense strand) were annealed (1:1 bymolar equivalents) at 90° C. for 3 min followed by RT 40 min) to producethe duplexes.

Example 13. siNA Activity Assays

This example provides exemplary methods for testing the activity of thesiNAs disclosed herein.

In Vitro Assay:

Homo sapiens HepG2.2.15 cells were cultured in Dulbecco's ModifiedEagle's Medium (DMEM) (ATCC 30-2002) supplemented to also contain 10%fetal calf serum (FCS). Cells were incubated at 37° C. in an atmospherewith 5% CO₂ in a humidified incubator. For transfection of HepG2.2.15cells with HBV targeting siRNAs, cells were seeded at a density of 15000cells/well in 96-well regular tissue culture plates. Transfection ofcells was carried out using RNAiMAX (Invitrogen/Life Technologies)according to the manufacturer's instructions. Dose-response experimentswere done with oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625,0.3125, 0.15625 and 0.07813 nM. For each HBV targeting siRNA treatment(e.g., ds-siRNA, as identified by the ds-siNA ID in Table 6), four wellswere transfected in parallel, and individual data points were collectedfrom each well. After 24 h of incubation with siRNA, media was removed,and cells were lysed and analyzed with a QuantiGene2.0 branched DNA(bDNA) probe set specific for HBV genotype D (also called Hepatitis Bvirus subtype ayw, complete genome of 3182 base-pairs) as present incell line HepG2.2.15.

For each well, the HBV on-target mRNA levels were normalized to theGAPDH mRNA level. As shown in Table 6, the activity of the HBV targetingds-siRNAs was expressed as EC50, 50% reduction of normalized HBV RNAlevel from no drug control. As shown in Table 6, the cytotoxicity of theHBV targeting ds-siRNAs was expressed by CC50 of 50% reduction of GAPDHmRNA from no drug control.

Unconjugated siRNA 1) with or without a phosphorylation blocker; and 2)with or without end caps (e.g., 5′-stabilized end cap) are transfectedinto in vitro disease models or in vitro toxicity models. Aftertransfection, target reduction and/or cell viability is measured andcompared after a period of incubation. For HBV, exemplary disease cellmodels include, but are not limited to, HepG2.2.15, HepG2.117 or liveHBV infected HepG2-NTCP or Primary Human Hepatocytes.

In Vivo Assay:

GalNAc conjugated siRNA 1) with or without phosphorylation blocker; and2) with or without 5′-end caps are dosed subcutaneously or intravenouslyin animal disease models. The target knockdown magnitude and duration ismeasured from serum or liver samples and compared to each other and/orcontrol animals (e.g., non-treated diseased animals). In some instances,the toxicity of the siRNAs is compared through routine Clinpath orHistopath assays. For HBV, exemplary animal efficacy models include, butare not limited to, AAV-HBV mouse model, HBV transgenic mouse model, PXBor FRG mouse models.

Example 14. Ds-siNA Testing in AAV-HBV Mouse Model

In this example, the efficacy of ds-siNAs in treating HBV in anadeno-associated virus (AAV)-HBV mouse model was evaluated. AAV-HBV micewere subcutaneously injected with a single dose of (a) 5 mL/kg ofvehicle; or (b) 5 mg/kg a ds-siNA at day 0. The sequences of the ds-siNAtested in this example are shown in Table 7.

FIG. 4 shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G03), ds-siNA-0165 (G04),ds-siNA-0163 (G05), or ds-siNA-0166 (G06). These results demonstratethat the ds-siNAs containing various patterns of 2′-fluoro nucleotidesand 2′-O-methyl nucleotides can effectively treated HBV.

TABLE 7  ds-siNA sequences tested in AAV-HBV mouse modelSense strand sequence Antisense strand sequence  ds-siNA ID  (5′-3′)(5′-3′) ds-siNA- mCpsmCpsfGmUmGmUfGfCfAmCmUf mUpsfGpsmAmAmGmCmGmAmAmGm0160 UmCmGmCmUfUmCmA-p-(PS)2- UmGmCfAmCmAmCmGmGpsmUpsmCGalNAc4 (SEQ ID NO: 600) (SEQ ID NO: 272) ds-siNA-mGpsmUpsfGmGmUmGfGfAfCmUmU mApsfUpsmUmGmAmGmAmGmAmA 0165fCmUmCmUmCfAmAmU-p-(PS)2- mGmUmCfCmAmCmCmAmCpsmGpsmGalNAc4 (SEQ ID NO: 601) A (SEQ ID NO: 292) ds-siNA-mGpsmCpsmUmGmCmUfAmUfGfCfC mApsfApsmGmAmAfGmAmUmGmAm 0163mUmCmAmUmCmUmUmCmUmU-p- GmGmCfAmUfAmGmCmAmGmCpsmA(PS)2-GalNAc4 (SEQ ID NO: 602) psmG (SEQ ID NO: 287) ds-siNA-mUpsmGpsfUmGmCmAfCfUmUmCm mApsfGpsmGmUmGmAmAmGmCmGm 0166GmCmUmUmCmAfCmCmU-p-(PS)2- AmAmGfUmGmCmAmCmApsmCpsmGGalNAc4 (SEQ ID NO: 603) (SEQ ID NO: 303)

Example 15. Ds-siNA Activity Assay and Testing in AAV-HBV Mouse

Model

This example investigates the in vitro and in vivo activity of ds-siNAs.The sequences of the ds-siNAs tested in this example are shown in Table8. As shown in Table 8, the ds-siNAs comprise a sense and antisensestrand comprising a mixture of 2′-fluoro and 2′-O-methyl nucleotides.The total number of 2′-fluoro nucleotides in the ds-siNAs are between6-8. The 2′-fluoro nucleotides may be at specific positions, such asnucleotide position 3, 5, 7, 8, 9, 10, 11, 12, and/or 17 from the 5′ endof the sense strand or 2, 5, 6, 8, 10, 14, 16, 17, and/or 18. The2′-fluoro nucleotides and 2′-O-methyl nucleotides might occur atspecific patterns on the antisense strand, such as an alternating 1:2 or1:3 pattern, wherein 1 nucleotide is a 2′-fluoro nucleotide and 2 or 3nucleotides are 2-O-methyl nucleotides.

In Vitro Activity Assay

Homo sapiens HepG2.2.15 cells were cultured in Dulbecco's ModifiedEagle's Medium (DMEM) (ATCC 30-2002) supplemented to also contain 10%fetal calf serum (FCS). Cells were incubated at 37° C. in an atmospherewith 5% CO₂ in a humidified incubator. For transfection of HepG2.2.15cells with HBV targeting siRNAs, cells were seeded at a density of 15000cells/well in 96-well regular tissue culture plates. Transfection ofcells was carried out using RNAiMAX (Invitrogen/Life Technologies)according to the manufacturer's instructions. Dose-response experimentswere done with oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625,0.3125, 0.15625 and 0.07813 nM. For each HBV targeting siRNA treatment(e.g., ds-siRNA, as identified by the ds-siNA ID in Table 8), four wellswere transfected in parallel, and individual data points were collectedfrom each well. After 24 h of incubation with siRNA, media was removed,and cells were lysed and analyzed with a QuantiGene2.0 branched DNA(bDNA) probe set specific for HBV genotype D (also called Hepatitis Bvirus subtype ayw, complete genome of 3182 base-pairs) as present incell line HepG2.2.15.

For each well, the HBV on-target mRNA levels were normalized to theGAPDH mRNA level. Table 8 shows the activity of the HBV targetingds-siRNAs expressed as EC50, which is 50% reduction of normalized HBVRNA level from no drug control, where A=EC50<0.5 nM; B=0.5 nM<EC50<1;and C=EC50>1.

In vivo testing in AAV-HBV mouse model.

AAV/HBV is a recombinant AAV carrying replicable HBV genome. Takingadvantage of the highly hepatotropic feature of genotype 8 AAV, the HBVgenome can be efficiently delivered to the mouse liver cells. Infectionof immune competent mouse with AAV/HBV can result in long term HBVviremia, which mimics chronic HBV infection in patients. The AAV/HBVmodel can be used to evaluate the in vivo activity of various types ofanti-HBV agents. Mice were infected with AAV-HBV on day −28 of thestudy. The test articles or negative control (PBS) were dosedsubcutaneously (unless specified otherwise) as single dose on days 0 at5 mg/kg. Serial blood collections were usually taken every 5 days on day0, 5, 10 and 15 etc. until the termination of studies. Serum HBV Santigen (HBsAg) was assayed through ELISA.

GalNAc conjugated ds-siNAs were further tested at a single dose of 5mg/kg at day 0 in the adeno-associated virus (AAV)-HBV mouse model. Theresulting nadir log₁₀ reduction in serum HBsAg is presented in Table 8,where X≥1 log₁₀ reduction in HBsAg, Y is 0.5-1 log₁₀ reduction in HBsAg,and Z is <0.5 log₁₀ reduction in HBsAg.

FIG. 5A shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0160 (G03). AAV-HBV mice weresubcutaneously injected with a single dose of 5 mL/kg of vehicle or 5mg/kg of ds-siNA-0160 on day 0.

FIG. 5B shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0160 (G15). AAV-HBV mice weresubcutaneously injected with a single dose of 5 mL/kg of vehicle or 5mg/kg of ds-siNA-0160 on day 0.

FIG. 5C shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0160 (G03). AAV-HBV mice weresubcutaneously injected with a single dose of 5 mL/kg of vehicle or 5mg/kg of each ds-siNA on day 0.

FIG. 5D shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G03), or ds-siNA-0109 (G09).AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kgof vehicle or 5 mg/kg of each ds-siNA on day 0.

FIGS. 5E-5F show a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0169 (G18). AAV-HBV mice weresubcutaneously injected with a single dose of 5 mL/kg of vehicle or 5mg/kg of ds-siNA-0169 on day 0.

FIG. 5G shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0169 (G04). AAV-HBV mice weresubcutaneously injected with a single dose of 5 mL/kg of vehicle or 5mg/kg of ds-siNA-0169 on day 0.

FIG. 5H shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01) or ds-siNA-0169 (G04). AAV-HBV mice weresubcutaneously injected with a single dose of 5 mL/kg of vehicle or 5mg/kg of each ds-siNA on day 0.

FIG. 5I shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0169 (G04) or ds-siNA-0147 (G08).AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kgof vehicle or 5 mg/kg of each ds-siNA on day 0.

FIG. 5J shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0166 (G06), or ds-siNA-0153 (G14).AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kgof vehicle or 5 mg/kg of each ds-siNA on day 0.

FIG. 5K shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0163 (G05), or ds-siNA-0119 (G13).AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kgof vehicle or 5 mg/kg of each ds-siNA on day 0.

These results demonstrate that ds-siNAs comprising combination of2′-fluoro nucleotides and 2′-O-methyl nucleotides can be used to targetHBV X and S gene sequences, which resulted in successful treatment ofHBV.

As exemplified by ds-siNA-0160 and ds-siNA-0165, ds-siNAs comprising (a)a sense strand comprising 19 nucleotides, wherein 6 nucleotides are2′-fluoro nucleotides and 13 nucleotides are 2′-O-methyl nucleotides;(b) an antisense strand comprising 21 nucleotides, wherein 2 nucleotidesare 2′-fluoro nucleotides and 19 nucleotides are 2′-O-methylnucleotides; and (c) a conjugated moiety, wherein the conjugated moietyis attached to the 3′ end of the sense strand, resulted in successfultreatment of HBV as evidenced by HBsAg reduction in serum. See FIGS. 4and 5A-5D, and Table 8. For ds-siNA-0160 and ds-siNA-0165, the 2′-fluoronucleotides were located at positions 3, 7-9, 12, and 17 from the 5′ endof the sense strand and at positions 2 and 14 from the 5′ end of theantisense strand.

As exemplified by ds-siNA-0166, ds-siNAs comprising (a) a sense strandcomprising 19 nucleotides, wherein 4 nucleotides are 2′-fluoronucleotides and 15 nucleotides are 2′-O-methyl nucleotides; (b) anantisense strand comprising 21 nucleotides, wherein 2 nucleotides are2′-fluoro nucleotides and 19 nucleotides are 2′-O-methyl nucleotides;and (c) a conjugated moiety, wherein the conjugated moiety is attachedto the 3′ end of the sense strand, resulted in successful treatment ofHBV as evidenced by HBsAg reduction in serum. See FIGS. 4 and 5J, andTable 8. For ds-siNA-0166, the 2′-fluoro nucleotides were located atpositions 3, 7, 8, and 17 from the 5′ end of the sense strand and atpositions 2 and 14 from the 5′ end of the antisense strand.

As exemplified by ds-siNA-0153, ds-siNAs comprising (a) a sense strandcomprising 19 nucleotides; (b) an antisense strand comprising 21nucleotides, wherein the nucleotides in the antisense strand comprise atleast two alternating 1:3 modification pattern, and wherein approximate1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methylnucleotides in repeat pattern; and (c) a conjugated moiety, wherein theconjugated moiety is attached to the 3′ end of the sense strand,resulted in successful treatment of HBV as evidenced by HBsAg reductionin serum. See FIG. 5J. For ds-siNA-0153, the sense strand comprises 62′-fluoro nucleotides at positions 3, 7-9, 12, and 17 from the 5′ end ofthe sense strand. In addition, the antisense strand comprises 5 repeatsof the 1:3 modification pattern starting at position 2 from the 5′ endof the antisense strand.

As exemplified by ds-siNA-0109, ds-siNAs comprising (a) a sense strandcomprising 19 nucleotides wherein 4 nucleotides are 2′-fluoronucleotides and 15 nucleotides are 2′-O-methyl nucleotides; (b) anantisense strand comprising 21 nucleotides, wherein 4 nucleotides are2′-fluoro nucleotides and 17 nucleotides are 2′-O-methyl nucleotides;and (c) a conjugated moiety, wherein the conjugated moiety is attachedto the 3′ end of the sense strand, resulted in successful treatment ofHBV as evidenced by HBsAg reduction in serum. See FIG. 5D. Fords-siNA-0109 the sense strand comprises 4 2′-fluoro nucleotides atpositions 5 and 7-9 from the 5′ end of the sense strand. In addition,the antisense strand comprises 5 repeats of the 1:2 modification patternstarting at positions 2, 5, 8, 14, and 17 from the 5′ end of theantisense strand.

As exemplified by ds-siNA-0147, ds-siNAs comprising (a) a sense strandcomprising 19 nucleotides; (b) an antisense strand comprising 21nucleotides, wherein the nucleotides in the antisense strand comprise atleast two alternating 1:2 modification pattern, and wherein approximate1 nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methylnucleotides in repeat pattern; and (c) a conjugated moiety, wherein theconjugated moiety is attached to the 3′ end of the sense strand,resulted in successful treatment of HBV as evidenced by HBsAg reductionin serum. See FIG. 5I. For ds-siNA-0147, the 2′-fluoro nucleotides werelocated at positions 5 and 7-9 from the 5′ end of the sense strand andat positions 2, 6, 14, and 16 from the 5′ end of the antisense strand.

TABLE 8  ds-siNA tested in AAV-HBV Mouse Model ds- EC50 HBsAg siNASense strand  Antisense strand  HepG2. Nadir ID sequence (5′-3′)sequence (5′-3′) 2.15* (Log)** ds- mCpsmCpsmGmUfGmUfGfCfmUpsfGpsmAmAfGmCmGfA siNA- AmCmUmUmCmGmCmUmU mAmGmUmGmCfAmCmAfC 0109mCmA-p-(PS)2-GalNac4 (SEQ mGmGpsmUpsmC (SEQ ID ID NO: 604) NO: 605) ds-mGpsmCpsmUmGfCmUmAm mApsfApsmGmAmAmGmA siNA- UfGfCfCmUmCfAmUmCmUmUmGmAmGmGmCfAmUm 0119 mUfCmUmU-p-(PS)2-GalNac4 AmGmCmAmGmCpsmApsm(SEQ ID NO: 606) G (SEQ ID NO: 495) ds- mGpsmUpsmGmGfUmGfGfAfmApsfUpsmUmGmAfGmAm siNA- CmUmUmCmUmCmUmCmA GmAmAmGmUmCfCmAfCm 0147mAmU-p-(PS)2-GalNac4 (SEQ CmAmCpsmGpsmA (SEQ ID ID NO: 607) NO: 608) ds-mUpsmGpsfUmGmCmAfCfUf mApsfGpsmGmUmGfAmAm siNA- UmCmGfCmUmUmCmAfCmGmCfGmAmAmGfUmGmC 0153 CmU-p-(PS)2-GalNac4 (SEQ mAfCmApsmCpsmG (SEQID NO: 609) ID NO: 610) ds- mGpsmCpsfGmGmGmGfUfUf mUpsfCpsmAmAmCmAmAmGmA X siNA- UmUmUfCmUmUmGmUfUm AmAmAmAmAfCmCmCmCmGm 0167GmA-p-(PS)2-GalNac4 (SEQ CpsmCpsmU ID NO: 611) (SEQ ID NO: 285) ds-mGpsmCpsfGmGmGmGfUfU mUpsfCpsmAmAmCmAmA C X siNA- mUmUmUmCmUmUmGmUfmGmAmAmAmAmAfCmCm 0162 UmGmA-p-(PS)2-GalNac4 CmCmGmCpsmCpsmU (SEQ(SEQ ID NO: 612) ID NO: 285) ds- mGpsmUpsfGmGmUmGfGfAfmApsfUpsmUmGmAmGmA A X siNA- CmUmUfCmUmCmUmCfAm mGmAmAmGmUmCfCmAm 0165AmU-p-(PS)2-GalNac4 (SEQ CmCmAmCpsmGpsmA (SEQ ID NO: 601) ID NO: 292)ds- mUpsmCpsmGmUmGmGfUm mApsfUpsmUmGmAfGmAm A X siNA-GfGfAfCmUmUmCmUmCmU GmAmAmGmUmCfCmAfCm 0168 mCmAmAmU-p-(PS)2-CmAmCmGmApsmGpsmU GalNac4 (SEQ ID NO: 613) (SEQ ID NO: 298) ds-mGpsmCpsmUmGmCmUfAm mApsfApsmGmAmAfGmAm A Y siNA- UfGfCfCmUmCmAmUmCmUUmGmAmGmGmCfAmUfA 0163 mUmCmUmU-p-(PS)2- mGmCmAmGmCpsmApsmGGalNac4 (SEQ ID NO: 602) (SEQ ID NO: 287) ds- mCpsmUpsfGmCmUmAfUfGfmApsfGpsmAmAmGmAmU A Y siNA- CmCmUfCmAmUmCmUfUm mGmAmGmGmCmAfUmAm 0161CmU-p-(PS)2-GalNac4 (SEQ GmCmAmGpsmCpsmA (SEQ ID NO: 614) ID NO: 277)ds- mCpsmCpsfGmUmGmUfGfCf mUpsfGpsmAmAmGmCmG A X siNA-AmCmUfUmCmGmCmUfUm mAmAmGmUmGmCfAmCm 0160 CmA-p-(PS)2-GalNac4 (SEQAmCmGmGpsmUpsmC (SEQ ID NO: 600) ID NO: 272) ds- mCpsmCpsfGmUmGmUfGfCfmUpsfGpsmAmAmGmCmG A X siNA- AmCmUfUmCmGmCmUfUm mAmAmGmUmGmCfAmCm 0169CmA-p-(PS)2-GalNac4 (SEQ AmCmGmGpsTpsT (SEQ ID ID NO: 600) NO: 375) ds-mUpsmGpsfUmGmCmAfCfUf mApsfGpsmGmUmGmAmAmGm A X siNA- UmCmGfCmUmUmCmAfCmCmGmAmAmGfUmGmCmAmCm 0170 CmU-p-(PS)2-GalNac4 (SEQ ApsmCpsmG ID NO: 609)(SEQ ID NO: 303) ds- mUpsmGpsfUmGmCmAfCfU mApsfGpsmGmUmGmAmA A X siNA-mUmCmGmCmUmUmCmAfC mGmCmGmAmAmGfUmGm 0166 mCmU-p-(PS)2-GalNAc4CmAmCmApsmCpsmG (SEQ (SEQ ID NO: 615) ID NO: 303) ds-mUpsmGpsfUmGmCmAfCfU mApsfGpsmGmUmGmAmAmGm A X siNA- mUmCmGmCmUmUmCmAfCCmGmAmAmGfUmGmCmAmCm 0171 mCmU-p-(PS)2-GalNac4 ApsTpsT (SEQ ID NO: 615)(SEQ ID NO: 407) mX = 2′-O-methyl nucleotide; fX = 2′-fluoro nucleotide;ps = phosphorothioate linkage *For EC50, A = EC50 < 0.5 nM; B = 0.5 nM< EC50 < 1; and C = EC50 > 1. **For HBsAg Nadir, X ≥ 1 log₁₀ reductionin HBsAg, Y is 0.5-1 log₁₀ reduction in HBsAg, and Z is < 0.5 log₁₀reduction in HBsAg.

Example 16. Testing of Ds-siNAs Having a 5′-Stabilized End Cap inAAV-HBV Mouse Model

This example investigates the in vivo activity of ds-siNAs having a5′-stabilized end cap. The sequences of the ds-siNAs tested in thisexample are shown in Table 9.

FIG. 6A shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G15) (ds-siNA without a5′-stabilized end cap, e.g., vinyl phosphonate), or ds-siNA-080 (G14)(ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate). AAV-HBVmice were subcutaneously injected with a single dose of 5 mL/kg ofvehicle or 5 mg/kg of each ds-siNA on day 0. The resulting nadir log₁₀reduction in serum HBsAg is presented in Table 9, where X≥1 log₁₀reduction in HBsAg, Y is 0.5-1 log₁₀ reduction in HBsAg, and Z is <0.5log₁₀ reduction in HBsAg.

FIG. 6B shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0169 (G16) (ds-siNA without a5′-stabilized end cap, e.g., vinyl phosphonate), or ds-siNA-081 (G13)(ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate). AAV-HBVmice were subcutaneously injected with a single dose of 5 mL/kg ofvehicle or 5 mg/kg of each ds-siNA on day 0. The resulting nadir log₁₀reduction in serum HBsAg is presented in Table 9, where X≥1 log₁₀reduction in HBsAg, Y is 0.5-1 log₁₀ reduction in HBsAg, and Z is <0.5log₁₀ reduction in HBsAg.

FIG. 7A shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0165 (G18) (ds-siNA without a5′-stabilized end cap, e.g., vinyl phosphonate), or ds-siNA-0127 (G17)(ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate). AAV-HBVmice were subcutaneously injected with a single dose of 5 mL/kg ofvehicle or 5 mg/kg of each ds-siNA on day 0. The resulting nadir log₁₀reduction in serum HBsAg is presented in Table 9, where X≥1 log₁₀reduction in HBsAg, Y is 0.5-1 log₁₀ reduction in HBsAg, and Z is <0.5log₁₀ reduction in HBsAg.

FIG. 7B shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0168 (G20) (ds-siNA without a5′-stabilized end cap, e.g., vinyl phosphonate), or ds-siNA-0150 (G19)(ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate). AAV-HBVmice were subcutaneously injected with a single dose of 5 mL/kg ofvehicle or 5 mg/kg of each ds-siNA on day 0. The resulting nadir log₁₀reduction in serum HBsAg is presented in Table 9, where X≥1 log₁₀reduction in HBsAg, Y is 0.5-1 log₁₀ reduction in HBsAg, and Z is <0.5log₁₀ reduction in HBsAg.

These results demonstrate that the addition of a 5′-stabilized end capcan improve the efficacy of ds-siNAs without a 5′-stabilized end cap.

TABLE 9  ds-siNA sequences and HBsAg Nadir HBsAg ds-siNA Sense strand Antisense strand  Nadir ID sequence (5′-3′) sequence (5′-3′) (Log)*ds-siNA- mCpsmCpsfGmUmGmUfGfC mUpsfGpsmAmAmGmCmGmAmAm Y 0160fAmCmUfUmCmGmCmUfU GmUmGmCfAmCmAmCmGmGpsm mCmA-(PS)2-p-GalNAc4UpsmC (SEQ ID NO: 272) (SEQ ID NO: 616) ds-siNA- mCpsmCpsfGmUmGmUfGfCvmUpsfGpsmAmAmGmCmGmAmA X 080 fAmCmUfUmCmGmCmUfU mGmUmGmCfAmCmAmCmGmGpsmCmA-(PS)2-p-GalNAc4 mUpsmC (SEQ ID NO: 462) (SEQ ID NO: 616) ds-siNA-mCpsmCpsfGmUmGmUfGfC mUpsfGpsmAmAmGmCmGmAmAm Y 0169 fAmCmUfUmCmGmCmUfUGmUmGmCfAmCmAmCmGmGpsTp mCmA-(PS)2-p-GalNAc4 sT (SEQ ID NO: 375)(SEQ ID NO: 616) ds-siNA- mCpsmCpsfGmUmGmUfGfC vmUpsfGpsmAmAmGmCmGmAmA X081 fAmCmUfUmCmGmCmUfU mGmUmGmCfAmCmAmCmGmGpsT mCmA-(PS)2-p-GalNAc4psT (SEQ ID NO: 463) (SEQ ID NO: 616) ds-siNA- mGpsmUpsfGmGmUmGfGfAmApsfUpsmUmGmAmGmAmGmAm X 0165 fCmUmUfCmUmCmUmCfAAmGmUmCfCmAmCmCmAmCpsmG mAmU-(PS)2-p-GalNAc4 psmA (SEQ ID NO: 292)(SEQ ID NO: 617) ds-siNA-  mGpsmUpsfGmGmUmGfGfA vmApsfUpsmUmGmAmGmAmGmAX 0127 fCmUmUfCmUmCmUmCfA mAmGmUmCfCmAmCmCmAmCpsm mAmU-(PS)2-p-GalNAc4GpsmA (SEQ ID NO: 503) (SEQ ID NO: 617) ds-siNA-  mUpsmCpsmGmUmGmGfUmmApsfUpsmUmGmAfGmAmGmAmA Y 0168 GfGfAfCmUmUmCmUmCmmGmUmCfCmAfCmCmAmCmGmAp UmCmAmAmU-(PS)2-p- smGpsmU (SEQ ID NO: 298)GalNAc4 (SEQ ID NO: 618) ds-siNA-  mUpsmCpsmGmUmGmGfUmvmApsfUpsmUmGmAfGmAmGmAm X 0150 GfGfAfCmUmUmCmUmCmAmGmUmCfCmAfCmCmAmCmGm UmCmAmAmU-(PS)2-p- ApsmGpsmU (SEQ ID NO: 523)GalNAc4 (SEQ ID NO: 618) mX = 2′-O-methyl nucleotide; fX = 2′-fluoronucleotide; ps = phosphorothioate linkage; VP = vinyl phosphonate *ForHBsAg Nadir, X ≥ 1 log₁₀ reduction in HBsAg, Y is 0.5-1 log₁₀ reductionin HBsAg, and Z is < 0.5 log₁₀ reduction in HBsAg.

Example 17. Efficacy of a Combination Therapy in AAV-HBV Mouse

Model

This example investigates the efficacy of a combination therapycomprising an antisense oligonucleotide (ASO 1, 5′Ga1NAc4-ps-Ga1NAc4-ps-Ga1NAc4-po-mA-po-lnGpslnApslnTpslnApslnApsApsAps(50H)CpsGpsg5m)Cpg(5 m)CpsGps(5 m)CpslnApslnG pslnApscp(5 m)C-3′(SEQ ID NO: 534)) anda ds-siNA-0160 for treating HBV in an AAV-HBV mouse model.

FIG. 8A shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G06), ASO 1 (G18), or acombination of ds-siNA-0160 and ASO 1 (G20). AAV-HBV mice weresubcutaneously injected with (a) 5 mL/kg of vehicle, three times a week,from days 0-42 (G01); (b) a single dose of 3 mg/kg of ds-siNA-0160 onday 0 (G06); (c) 3 mg/kg of ASO 1 on a weekly basis, from days 0-21(G18); or (d) a combination of ASO 1 and ds-siNA-0160, wherein ASO 1 wasadministered at a dose of 3 mg/kg on a weekly basis, from days 0-21; andds-siNA-0160 was administered as a single dose of 3 mg/kg at day 0.

FIG. 8B shows a graph of the change in serum HBsAg from AAV-HBV micetreated with vehicle (G01), ds-siNA-0160 (G06), ASO 1 (G18), or acombination of ds-siNA-0160 and ASO 1 (G20). AAV-HBV mice weresubcutaneously injected with (a) 5 mL/kg of vehicle, three times a week,from days 0-42 (G01); (b) a single dose of 10 mg/kg of ds-siNA-0160 onday 0 (G06); (c) 10 mg/kg of ASO 1 on a weekly basis, from days 0-21(G18); or (d) a combination of ASO 1 and ds-siNA-0160, wherein ASO 1 wasadministered at a dose of 10 mg/kg on a weekly basis, from days 0-21;and ds-siNA-0160 was administered as a single dose of 3 mg/kg at day 0.

FIG. 8C shows a graph of a synergy analysis of an in vitro combinationtherapy with the ASO 2 and ds-siNA-0164. For the ds-siNA-0164combination studies with ASO 2, 35,000 cells per well were reversetransfected in a collagen I-coated 96-well plate (Corning, Biocoat;Catalog 356698). Test articles ds-siNA-0164 and ASO 2 were diluted inOpti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific; Catalog31985088) to 40x the desired final test concentration then seriallydiluted (1:3) up to 5 or 9 distinct concentrations, respectively. A 3.25μL aliquot of each diluted compound was combined in a checkerboardfashion. This combination of compounds was mixed with 0.3 μLLipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific,Catalog 13778150) and 6.2 μL of Opti-MEM™ I Reduced Serum Medium. Afterincubating for 20 minutes, the mixture was added to the cells. Space wasalso allotted for titrations of each compound alone as referencecontrols. Cells were incubated with compounds for 3 days at 37° C. in a5% CO₂ atmosphere. After that, HBsAg in the supernatant of cell culturewas assayed by ELISA and cell viability was measured with Cell TiterGlow, the same procedures as in HepG2.2.15 in vitro assay section. TheHBsAg reduction synergy between two test articles were analyzed usingMacSynergy Software.

These results demonstrate that a combination therapy with ASO 1 andds-siNA-0160 resulted in a greater reduction in serum HBsAg as comparedto treatment with ASO 1 or ds-siNA-0160 alone.

Example 18. siNA Activity Assays

This example evaluates the activity of the siNAs disclosed in Table 10(as identified by the ds-siNA ID). siRNAs were synthesized as describedin Example 1. A conjugated moiety (e.g., ligand monomer) was furtherconjugated to the 3′ end of the sense strand (note: for ds-siNA-067 andds-siNA-083, the ligand monomer was conjugated to the 5′ end of thesense strand). A 5′-stabilized end cap was further attached to the 5′end of the antisense strand of some siRNAs.

In Vitro Assay:

Homo sapiens HepG2.2.15 cells were cultured in Dulbecco's ModifiedEagle's Medium (DMEM) (ATCC 30-2002) supplemented to also contain 10%fetal calf serum (FCS). Cells were incubated at 37° C. in an atmospherewith 5% CO₂ in a humidified incubator. For transfection of HepG2.2.15cells with HBV targeting siRNAs, cells were seeded at a density of 15000cells/well in 96-well regular tissue culture plates. Transfection ofcells was carried out using RNAiMAX (Invitrogen/Life Technologies)according to the manufacturer's instructions. Dose-response experimentswere done with oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625,0.3125, 0.15625 and 0.07813 nM. For each HBV targeting siRNA treatment(e.g., ds-siRNA, as identified by the ds-siNA ID in Table 6), four wellswere transfected in parallel, and individual data points were collectedfrom each well. After 24 h of incubation with siRNA, media was removed,and cells were lysed and analyzed with a QuantiGene2.0 branched DNA(bDNA) probe set specific for HBV genotype D (also called Hepatitis Bvirus subtype ayw, complete genome of 3182 base-pairs) as present incell line HepG2.2.15.

For each well, the HBV on-target mRNA levels were normalized to theGAPDH mRNA level. As shown in Table 10, the activity of the HBVtargeting ds-siRNAs was expressed as EC50, 50% reduction of normalizedHBV RNA level from no drug control, where A=EC50<5 nM; B=5 nM<EC50<10;C=EC50≥10. As shown in Table 10, the cytotoxicity of the HBV targetingds-siRNAs was expressed by CC50 of 50% reduction of GAPDH mRNA from nodrug control.

In Vivo Assay:

GalNAc conjugated siRNA with or without 5′-stabilized end caps weresubcutaneously injected at a single dose of 5 mg/kg into AAV-HBV mice.The target knockdown magnitude was measured from serum. The resultingmax HBsAg knockdown (log₁₀) is presented in Table 10, where X≥1 log₁₀reduction in HBsAg, Y is 0.5-1 log₁₀ reduction in HBsAg, and Z is <0.5log₁₀ reduction in HBsAg.

Example 19: Analysis of 5′-Stabilized End Cap on the Efficacy of siNAs

In this example, the role of a 5′-stabilized end cap on the efficacy ofsiNAs was investigated. Specifically, the first nucleotide on the 5′ endof the antisense strand was modified to contain a 5′-stabilized end cap.The ds-siNAs investigated in this example are shown in the table below:

ds-siNA Sense Strand  Antisense Strand Sequence ID Sequence (5′→3′)(5′→3′) ds-siNA- mUpsmGpsfUmGmCmAfCfUmUmCmG mApsfGpsmGmUmGmAmAmGmCm 0166mCmUmUmCmAfCmCmU-p-(PS)2- GmAmAmGfUmGmCmAmCmApsmGalNAc4 (SEQ ID NO: 615) CpsmG (SEQ ID NO: 303) ds-siNA-mUpsmGpsfUmGmCmAfCfUmUmCmG vmApsfGpsmGmUmGmAmAmGmC 0155mCmUmUmCmAfCmCmU-p-(PS)2- mGmAmAmGfUmGmCmAmCmApsGalNAc4 (SEQ ID NO: 615) mCpsmG (SEQ ID NO: 525) ds-siNA-mUpsmGpsfUmGmCmAfCfUmUmCmG d2vmApsfGpsmGmUmGmAmAmG 0157mCmUmUmCmAfCmCmU-p-(PS)2- mCmGmAmAmGfUmGmCmAmCm GalNAc4 (SEQ ID NO: 615)ApsmCpsmG (SEQ ID NO: 529) mX = 2′-O-methyl nucleotide; fX = 2′-fluoronucleotide; vmA = 5′-vinyl phosphonate 2′-O-methyl adenosine; d2vmA= deuterated 5′ vinyl phosphonate adenosine; ps = phosphorothioatelinkage

AAV-HBV mice were subcutaneously injected with vehicle or ds-siNAs.ds-siNA-0166, ds-siNA-0155, or ds-siNA-0157 were subcutaneously injectedat a single dose of 5 mg/kg into AAV-HBV mice. The target knockdownmagnitude is measured from serum. As shown in FIG. 9 , the presence ofthe 5′ stabilized end cap in the first nucleotide from the 5′ end of theantisense strand in ds-siNA-0155 (triangle) and ds-siNA-0157 (square)improved the efficacy of the siNA (squares and triangles) as compared tothe siNA without the 5′ stabilized end cap (ds-siNA-0166, diamond). Inaddition, the presence of the deuterated 5′ vinyl phosphonate inds-siNA-0157 resulted in a greater improvement in efficacy of a ds-siNAas compared to the presence of the 5′ vinylphosphanate in ds-siNA-0155.These results demonstrate that a 5′ stabilized end cap improves theefficacy of siNAs, with the greatest improvement seen in siNAscontaining deuterated 5′ vinyl phosphonate.

Example 20: Analysis of HBV siRNA S and X Combination Therapy

In this example, combination therapy using an siNA targeting the S geneof HBV and an siNA targeting the X gene of HBV was examined. AAV-HBVmice were treated with vehicle, a single siNA therapy, or a combinationsiNA therapy targeting the S gene and X gene of HBV. AAV-HBV mice weresubcutaneously injected with a single dose of ds-siNA-0160 ords-siNA-0165 on day 0. For the combination siNA therapy, AAV-HBV micewere subcutaneously injected with a single dose of 1.5 mg/kg ofds-siNA-0165 (S trigger) and 1.5 mg/kg of ds-siNA-0160 (X trigger) onday 0. As shown in FIG. 10 , the combination therapy with a siNAtargeting the S gene and a siNA targeting the X gene was more potentthan the single therapy with ds-siNA-0165 or ds-siNA-0160.

Example 21. Synthesis of Monomer

Preparation of (2a): To a solution of 1a (10.0 g, 29.5 mmol) in ACN(200.0 mL), KSAc (13.5 g, 118.6 mmol) was added at r.t., the mixture wasstirred at r.t. for 15 h, TLC showed 1a was consumed completely. Mixturewas filtered by silica gel and filter cake was washed with DCM (100.0mL), the filtrate was concentrated to give crude 2a (11.1 g) as an oil.¹H-NMR (400 MHz, CDCl₃): S 7.32-7.24 (m, 5H), 7.16 (d, J=8.9 Hz, 4H),6.82 (d, J=8.9 Hz, 4H), 3.82 (s, 6H), 2.28 (s, 3H).

Preparation of (3a): To a solution of crude 2a (11.1 g, 29.2 mmol) inTHF (290.0 mL), LiAlH₄ (2.0 g, 52.6 mmol) was added at 0° C. and keptfor 10 min, reaction was stirred at r.t. for 5 h under N₂, TLC showed 2awas consumed completely. Mixture was put into aqueous NaHCO₃ solutionand extracted with EA (500.0 mL*2), organic phase was concentrated togive crude which was purified by column chromatography (SiO₂, PE/EA=30:1to 10:1) to give 3a (8.1 g, 95% purity) as a white solid. ESI-LCMS: m/z335.3 [M−H]⁻; ¹H-NMR (400 MHz, CDCl₃): S 7.33-7.24 (m, 5H), 7.19 (d,J=8.8 Hz, 4H), 6.82 (d, J=8.8 Hz, 4H), 3.83 (s, 6H), 3.09 (s, 1H).

Preparation of (2): To a solution of 1(20.0 g, 81.3 mmol) in pyridine(400.0 mL), MsCl (10.23 g, 89.43 mmol) was added dropwise at −10° C.,reaction was stirred at −10° C. for 1 h, LCMS showed 1 was consumedcompletely, 100.0 mL aqueous NaHCO₃ solution was added and extractedwith DCM (100.0 mL*2), organic phase was concentrated to give crudewhich was purified by column chromatography (SiO₂, DCM/MeOH=30:1 to10:1) to give 2 (9.5 g, 97% purity) as a white solid. ESI-LCMS: m/z325.3 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.45 (s, 1H), 7.64-7.62 (d,J=8.0 Hz, 1H), 5.92-5.85 (m, 2H), 5.65-5.63 (d, J=8.0 Hz, 1H), 5.26-5.11(m, 1H), 4.53-4.37 (m, 2H), 4.27-4.16 (m, 1H), 4.10-4.04 (m, 1H), 3.23(s, 3H).

Preparation of (3): Intermediate 3 was prepared by prepared according toreaction condition described in reference Helvetica Chimica Acta, 2004,87. 2812. To a solution of 2 (9.2 g, 28.3 mmol) in dry DMSO (130.0 mL).DMTrSH (14.31 g, 42.5 mmol) was added, followed by tetramethylguanidine(3.6 g, 31.2 mmol) was added under N₂, reaction was stirred at r.t. for3 h, LCMS showed 2 was consumed completely. 100.0 mL H₂O was added andextracted with EA (100.0 mL*2), organic phase was concentrated to givecrude which was purified by column chromatography (SiO₂, PE/EA=5:1 to1:1) to give 3 (12.0 g, 97% purity) as a white solid. ESI-LCMS: m/z563.2 [M−H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ 11.43-11.42 (d, J=4.0 Hz,1H), 7.57-7.55 (d, J=8.0 Hz, 1H), 7.33-7.17 (m, 9H), 6.89-6.86 (m, 4H),5.80-5.74 (m, 1H), 5.65-5.62 (m, 1H), 5.58-5.57 (d, J=4.0 Hz, 1H),5.16-5.01 (m, 1H), 3.98-3.90 (m, 1H), 3.73 (s, 6H), 3.73-3.67 (m, 1H),2.50-2.37 (m, 2H).

Preparation of Example 21 monomer: To a solution of 3 (10.0 g, 17.7mmol) in dichloromethane (120.0 mL) with an inert atmosphere of nitrogenwas added CEOP[N(iPr)₂]₂ (6.4 g, 21.2 mmol) and DCI (1.8 g, 15.9 mmol)in order at room temperature. The resulting solution was stirred for 1.0h at room temperature and diluted with 50 mL dichloromethane and washedwith 2×50 mL of saturated aqueous sodium bicarbonate and 1×50 mL ofsaturated aqueous sodium chloride respectively. The organic phase wasdried over anhydrous sodium sulfate, filtered and concentrated till noresidual solvent left under reduced pressure. The residue was purifiedby Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=6/1; Detector, UV 254 nm. Thisresulted in to give Example 21 monomer (12.8 g, 98% purity, 93% yield)as an oil. ESI-LCMS: m/z 765.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ11.44 (s, 1H), 7.70-7.66 (m, 1H), 7.32-7.18 (m, 9H), 6.89-6.85 (m, 4H),5.80-5.64 (m, 2H), 5.38-5.22 (m, 1H), 4.38-4.15 (m, 1H), 3.81-3.70 (m,8H), 3.61-3.43 (m, 3H), 2.76-2.73 (m, 1H), 2.66-2.63 (m, 1H), 2.50-2.41(m, 2H), 1.12-1.05 (m, 9H), 0.97-0.95 (m, 3H); ³¹P-NMR (162 MHz,DMSO-d6): δ 149.01, 148.97, 148.74, 148.67; ¹⁹F-NMR (376 MHz, DMSO-d6):δ 149.01, 148.97, 148.74, 148.67.

Example 22. Synthesis of Monomer

Preparation of (2): To a stirred solution of 1 (2.0 g, 8.8 mmol) inpyridine (20 mL) were added DMTrCI (3.3 g, 9.7 mmol) at r.t. Thereaction mixture was stirred at r.t. for 2.5 hrs. With ice-bath cooling,the reaction was quenched with water and the product was extracted withEA (100 mL). The organic phase was evaporated to dryness under reducedpressure to give a residue which was purified by silica gel columnchromatography (eluent, DCM: MeOH=50:1˜20:1) to give 2 (3.7 g, 7.2 mmol,80.1%) as a white solid. ESI-LCMS: m/z 527 [M−H]⁻.

Preparation of (3): To the solution of 2 (2.8 g, 5.3 mmol) in dry DMF(56 mL) was added (CD30)₂Mg (2.9 g, 31.8 mmol) at r.t. under N₂atmosphere. The reaction mixture was stirred at 100° C. for 15 hrs. Withice-bath cooling, the reaction was quenched with saturated aq. NH₄Cl andextracted with EA (300 mL). The combined organic layer was washed withwater and brine, dried over Na₂SO₄, and concentrated to give a residuewhich was purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1;Detector, UV 254 nm. This resulted in to give 3 (2.0 g, 3.6 mmol, 67.9%)as a white solid. ESI-LCMS: m/z 562 [M−H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ11.38 (s, 1H), 7.73 (d, J=8 Hz, 1H), 7.46-7.19 (m, 9H), 6.91 (d, J=7.4Hz, 4H), 5.81-5.76 (AB, J=20 Hz, 1H), 5.30 (d, J=8 Hz, 1H), 5.22 (s,1H), 4.25-4.15 (m, 1H), 3.99-3.92 (m, 1H), 3.85-3.79 (m, 1H), 3.74 (s,6H), 3.34-3.18 (m, 31H).

Preparation of Example 22 monomer: To a suspension of 3 (2.0 g, 3.5mmol) in DCM (20 mL) was added DCI (357 mg, 3.0 mmol) and CEP[N(iPr)₂]₂(1.3 g, 4.3 mmol). The mixture was stirred at r.t. for 1 h. LC-MS showed3 was consumed completely. The solution was washed with water twice andwashed with brine and dried over Na₂SO₄. Then concentrated to give aresidue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 22monomer (2.1 g, 2.7 mmol, 77.1%) as a white solid. ESI-LCMS: m/z 764[M+H]⁺; ¹H-NMR (400 MHz, ACN-d3): δ 9.45-8.90 (m, 1H, exchanged withD20), 7.88-7.66 (m, 1H), 7.50-7.18 (m, 9H), 6.93-6.80 (m, 4H), 5.85 (d,J=8.2 Hz, 1H), 5.29-5.16 (m, 1H), 4.57-4.37 (m, 1H), 4.18-4.09 (m, 1H),3.98-3.90 (m, 1H), 3.90-3.74 (m, 7H), 3.74-3.50 (m, 3H), 3.48-3.31 (m,2H), 2.70-2.61 (m, 1H), 2.56-2.46 (m, 1H), 1.24-1.12 (m, 9H), 1.09-0.99(m, 3H). ³¹P-NMR (162 MHz, ACN-d3): 6=149.87, 149.55.

Example 23. Synthesis of Monomer

Preparation of (2): To the solution of 1 (39.2 g, 151.9 mmol) in DMF(390.0 mL) was added imidazole (33.0 g, 485.3 mmol) and TBSCl (57.2 g,379.6 mmol) at 0° C. The reaction mixture was stirred at roomtemperature for 15 hrs under N₂ atmosphere. After addition of water, theresulting mixture was extracted with EA (500.0 mL). The combined organiclayer was washed with water and brine, dried over Na₂SO₄, concentratedto give the crude 2 (85.6 g) as a white solid which was used directlyfor next step. ESI-LCMS: m/z 487.7 [M+H]⁺.

Preparation of (3): A solution of crude 2 (85.6 g) in a mixture solventof TFA/H₂O=1/1 (400.0 mL) and THF (400.0 mL) was stirred at 0° C. for 30min. After completion of reaction, the resulting mixture was addedcon.NH₃*H₂O to pH=7, and then extracted with EA (500.0 mL). The organiclayer was washed with brine, dried over sodium sulfate and removed togive the residue was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=3/2within 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1; Detector, UV 254 nm. This resulted in to give 3 (36.6 g,98.4 mmol, 64.7% over two step) as a white solid. ESI-LCMS: m/z 372.5[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.36 (d, J=1 Hz, 1H), 7.92 (d, J=8Hz, 1H), 5.83 (d, J=5 Hz, 1H), 5.67-5.65 (m, 1H), 5.19 (s, 1H), 4.30 (t,J=5 Hz, 1H), 3.85-3.83 (m, 2H), 3.68-3.52 (m, 2H), 0.88 (s, 9H), 0.09(s, 6H).

Preparation of (4): To the solution of 3 (36.6 g, 98.4 mmol) in dry DCM(200.0 mL) and DMF (50.0 mL) was added PDC (73.9 g, 196.7 mmol),tert-butyl alcohol (188.0 mL) and Ac₂O (93.0 mL) at r.t under N₂atmosphere, the reaction mixture was stirred at r.t for 2 hrs. Thesolvent was removed to give a residue which was purified by silica gelcolumn chromatography (eluent, PE/EA=4:1-2:1) to give a residue whichwas purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give 4 (24.3 g, 54.9 mmol,55.8%) as a white solid. ESI-LCMS: m/z 443.2 [M+H]⁺; ¹H-NMR (400 MHz,DMSO-d6): δ 11.30 (d, J=1 Hz, 1H), 7.92 (d, J=8 Hz, 1H), 5.86 (d, J=6Hz, 1H), 5.67-5.65 (m, 1H), 4.33-4.31 (m, 1H), 4.13 (d, J=3 Hz, 1H),3.73-3.70 (m, 1H), 1.34 (s, 9H), 0.77 (s, 9H), 0.08 (s, 6H).

Preparation of (5): To the solution of 4 (18.0 g, 40.7 mmol) in dryTHF/MeOD/D20=10/2/1 (145.0 mL) was added NaBD₄ (5.1 g, 122.1 mmol) threetimes during an hour at 50° C., the reaction mixture was stirred at r.t.for 2 hrs. After completion of reaction, adjusted pH value to 7 withCH₃COOD, after addition of water, the resulting mixture was extractedwith EA (300.0 mL). The combined organic layer was washed with water andbrine, dried over Na₂SO₄, concentrated to give a residue which waspurified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1;Detector, UV 254 nm. This resulted in to give 5 (10.4 g, 27.8 mmol,68.3%) as a white solid. ESI-LCMS: m/z 375.2 [M+H]⁺; ¹H-NMR (400 MHz,DMSO-d6): δ 11.36 (d, J=1 Hz, 1H), 7.92 (d, J=8 Hz, 1H), 5.83 (d, J=5Hz, 1H), 5.67-5.65 (m, 1H), 5.19 (s, 1H), 4.30 (t, J=5 Hz, 1H),3.85-3.83 (m, 2H), 0.88 (s, 9H), 0.09 (s, 6H).

Preparation of (6): To a stirred solution of 5 (10.4 g, 27.8 mmol) inpyridine (100.0 mL) was added DMTrC1 (12.2 g, 36.1 mmol) at r.t., Thereaction mixture was stirred at r.t. for 2.5 hrs, the reaction wasquenched with water and extracted with EA (200.0 mL). The organic phasewas evaporated to dryness under reduced pressure to give a residue whichwas purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give 6 (13.5 g, 19.9 mmol,71.6%) as a white solid. ESI-LCMS: m/z 677.8 [M+H]⁺; ¹H-NMR (400 MHz,DMSO-d6): δ 11.39 (d, J=1 Hz, 1H), 7.86 (d, J=4 Hz, 1H), 7.35-7.21 (m,9H), 6.90-6.88 (m, 4H), 5.78 (d, J=2 Hz, 1H), 5.30-5.27 (m, 1H),4.33-4.30 (m, 1H), 3.91 (d, J=7 Hz, 1H), 3.85-3.83 (m, 1H), 3.73 (s,6H), 3.38 (s, 3H), 0.77 (s, 9H), 0.03 (s, 3H), 0.01 (s, 3H).

Preparation of (7): To a solution of 6 (13.5 g, 19.9 mmol) in THF (130.0mL) was added 1 M TBAF solution (19.0 mL). The reaction mixture wasstirred at r.t. for 1.5 hrs. LC-MS showed 6 was consumed completely.Water (500.0 mL) was added and extracted with EA (300.0 mL), the organiclayer was washed with brine and dried over Na₂SO₄. Then the organiclayer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 7 (10.9 g, 19.4 mmol, 97.5%) as a white solid.ESI-LCMS: m/z 563.6 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.39 (s, 1H),7.23 (d, J=8 Hz, 1H), 7.73 (d, J=8 Hz, 1H), 7.36-7.23 (m, 9H), 6.90 (d,J=8 Hz, 4H), 5.81 (d, J=3 Hz, 1H), 5.30-5.28 (m, 1H), 5.22 (d, J=7 Hz,1H), 4.20 (q, J=7 Hz, 1H), 3.93 (d, J=7 Hz, 1H), 3.81 (t, J=5 Hz, 1H),3.74 (s, 6H), 3.41 (s, 3H).

Preparation of Example 23 monomer: To a suspension of 7 (10.9 g, 19.4mmol) in DCM (100.0 mL) was added DCI (1.8 g, 15.7 mmol) andCEP[N(iPr)₂]₂ (6.1 g, 20.4 mmol). The mixture was stirred at r.t. for 1h. LC-MS showed 7 was consumed completely. The mixture was washed withwater twice and brine, dried over Na₂SO₄. Then concentrated to give aresidue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 23monomer (12.5 g, 14.5 mmol, 74.7%) as a white solid. ESI-LCMS: m/z 863.6[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.39 (s, 1H), 7.81-7.55 (m, 1H),7.40-7.22 (m, 9H), 6.92-6.87 (m, 4H), 5.83-5.80 (m, 1H), 5.32-5.25 (m,1H), 4.46-4.34 (m, 1H), 4.10-3.98 (m, 2H), 3.84-3.73 (m, 7H), 3.60-3.50(m, 3H), 3.42, 3.40 (s, 3H), 2.78 (t, J=6 Hz, 1H), 2.62-2.59 (m, 1H),2.07 (s, 1H), 1.17-0.96 (m, 12H); ³¹P-NMR (162 MHz, DMSO-d6): δ 149.37,149.06.

Example 24. Synthesis of Monomer

Preparation of (2): To the solution of 1 (13.0 g, 52.8 mmol) in DMF (100mL) was added imidazole (12.6 g, 184.8 mmol) and TBSCl (19.8 g, 132.0mmol) at 0° C., and the reaction mixture was stirred at room temperaturefor 15 h under N₂ atmosphere. After addition of water, the resultingproduct was extracted with EA (500 mL). The combined organic layer waswashed with water and brine, dried over Na₂SO₄, and concentrated to givethe crude 2 (30.6 g) as a white solid which was used directly for nextstep. ESI-LCMS: m/z 475 [M+H]⁺. WO2017106710A1

Preparation of (3): A solution of crude 2 (30.6 g) in a mixture solventof TFA/H₂O=1/1 (100 mL) and THF (100 mL) was stirred at 0° C. for 30min. After completion of reaction, the resulting mixture was addedcon.NH₃*H₂O to pH=7.5, and then the mixture was extracted with EA (500mL), the organic layer was washed with brine, dried over Na₂SO₄ andremoved to give the residue was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=3/2 within 20 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. This resulted in togive 3 (12.0 g, 33.3 mmol, 65.8% over two step) as a white solid.ESI-LCMS: m/z 361 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.39 (s, J=1 Hz,1H, exchanged with D20), 7.88 (d, J=8 Hz, 1H), 5.91-5.86 (m, 1H),5.66-5.62 (m, 1H), 5.21 (t, J=5.2 Hz, 1H, exchanged with D20), 5.18-5.03(m, 1H), 4.37-4.29 (m, 1H), 3.87-3.83 (m, 1H), 3.78-3.73 (m, 1H),3.56-3.51 (m, 1H), 0.87 (s, 9H), 0.09 (s, 6H). WO2017106710A1.

Preparation of (4): To the solution of 3 (11.0 g, 30.5 mmol) in dry DCM(60 mL) and DMF (15 mL) was added PDC (21. g, 61.0 mmol), tert-butylalcohol (45 mL) and Ac₂O (32 mL) at r.t under N₂ atmosphere. And thereaction mixture was stirred at r.t for 2 h. The solvent was removed togive a residue which was purified by silica gel column chromatography(eluent, PE: EA=4:1-2:1) to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give 4 (9.5 g, 22.0 mmol, 72.3%) as a white solid.ESI-LCMS: m/z 431 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.45 (s, J=1 Hz,1H, exchanged with D20), 7.93 (d, J=8.5 Hz, 1H), 6.02-5.97 (m, 1H),5.76-5.74 (m, 1H), 5.29-5.14 (m, 1H), 4.59-4.52 (m, 1H), 4.29-4.27 (m,1H), 1.46 (s, 9H), 0.89 (s, 9H), 0.12 (s, 6H).

Preparation of (5): To the solution of 4 (8.5 g, 19.7 mmol) in dryTHF/MeOD/D20=10/2/1 (80 mL) was added NaBD₄ (2.5 g, 59.1 mmol) threetimes per an hour at 50° C. And the reaction mixture was stirred at r.tfor 2 h. After completion of reaction, adjusted pH value to 7 withCH₃COOD, after addition of water, the resulting mixture was extractedwith EA (300 mL). The combined organic layer was washed with water andbrine, dried over Na₂SO₄, and concentrated to give a residue which waspurified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1;Detector, UV 254 nm. This resulted in to give 5 (3.5 g, 9.7 mmol, 50.3%)as a white solid. ESI-LCMS: m/z 363 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ11.41 (s, J=1 Hz, 1H, exchanged with D20), 7.88 (d, J=8 Hz, 1H),5.91-5.86 (m, 1H), 5.66-5.62 (m, 1H), 5.19 (t, J=5.2 Hz, 1H, exchangedwith D20), 5.18-5.03 (m, 1H), 4.37-4.29 (m, 1H), 3.87-3.83 (m, 1H), 0.88(s, 9H), 0.10 (s, 6H).

Preparation of (6): To a stirred solution of 5 (3.4 g, 9.7 mmol) inpyridine (35 mL) were added DMTrCl (3.4 g, 10.1 mmol) at r.t. And thereaction mixture was stirred at r.t for 2.5 h. With ice-bath cooling,the reaction was quenched with water and the product was extracted withEA (200 mL). The organic phase was evaporated to dryness under reducedpressure to give a residue which was purified by Flash-Prep-HPLC withthe following conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 20 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in togive 6 (PCT Int. Appl., 2019173602), (5.5 g, 8.3 mmol, 85.3%) as a whitesolid. ESI-LCMS: m/z 665 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.50 (d,J=1 Hz, 1H, exchanged with D20), 7.92 (d, J=4 Hz, 1H), 7.44-7.27 (m,9H), 6.96-6.93 (m, 4H), 5.94 (d, J=20.5 Hz, 1H), 5.39-5.37 (m, 1H),5.32-5.17 (m, 1H), 4.60-4.51 (m, 1H), 4.01 (d, J=8.8 Hz, 1H), 3.80 (s,6H), 0.80 (s, 9H), 0.09 (s, 3H), −0.05 (s, 3H).

Preparation of (7): To a solution of 6 (5.5 g, 8.3 mmol) in THF (50 mL)was added 1 M TBAF solution (9 mL). The reaction mixture was stirred atr.t. for 1.5 h. LC-MS showed 6 was consumed completely. Water (500 mL)was added. The product was extracted with EA (300 mL) and the organiclayer was washed with brine and dried over Na₂SO₄. Then the organiclayer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 7 (4.1 g, 7.5 mmol, 90.0%) as a white solid.ESI-LCMS: m/z 551 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.42 (s, 1H,exchanged with D20), 7.76 (d, J=8.2 Hz, 1H), 7.39-7.22 (m, 9H),6.90-6.88 (m, 4H), 5.83 (d, J=20.5 Hz, 1H), 5.65 (d, J=7.0 Hz, 1H,exchanged with D20), 5.29 (d, J=7.2 Hz, 1H), 5.18-5.03 (m, 1H),4.40-4.28 (m, 1H), 4.01 (d, J=8.8 Hz, 1H), 3.74 (s, 6H).

Preparation of Example 24 monomer: To a suspension of 7 (4.1 g, 7.5mmol) in DCM (40 mL) was added DCI (0.7 g, 6.4 mmol) and CEP[N(iPr)₂]₂(2.9 g, 9.7 mmol). The mixture was stirred at r.t. for 1 h. LC-MS showed7 was consumed completely. The solution was washed with water twice andwashed with brine and dried over Na₂SO₄. Then concentrated to give aresidue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 24monomer (5.0 g, 6.6 mmol, 90.0%) as a white solid. ESI-LCMS: m/z 751[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.43 (s, 1H), 7.85-7.82 (m, 1H),7.40-7.23 (m, 9H), 6.90-6.85 (m, 4H), 5.94-5.86 (m, 1H), 5.40-5.24 (m,2H), 4.74-4.49 (m, 1H), 4.12-4.09 (m, 2H), 3.79-3.47 (m, 10H), 2.78-2.59(m, 2H), 1.14-0.93 (m, 12H). ³¹P-NMR (162 MHz, DMSO-d6): δ 149.67,149.61, 149.32, 149.27.

Example 25. Synthesis of Monomer

Preparation of (4): To the solution of 3 (14.3 g, 25.4 mmol, Scheme 2)in pyridine (150 mL) was added imidazole (4.5 g, 66.6 mmol) and TBSCI(6.0 g, 40.0 mmol) at 0° C., and the reaction mixture was stirred atroom temperature for 15 h under N₂ atmosphere. After addition of water,the resulting mixture was extracted with EA (500 mL). The combinedorganic layer was washed with water and brine, dried over Na₂SO₄, andconcentrated to give the crude 4 (18.0 g) as a white solid which wasused directly for next step. ESI-LCMS: m/z 676 [M−H]⁻.

Preparation of (5): To the solution of crude 4 (18.0 g) in the solutionof DCA (6%) in DCM (200 mL) was added TES (50 mL) at r.t, and thereaction mixture was stirred at room temperature for 5-10 min. Aftercompletion of reaction, the resulting mixture was added pyridine topH=7, and then the solvent was removed and the residue was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 5 (6.5 g, 17.2 mmol, 67.7% for two step) as a whitesolid. ESI-LCMS: m/z 376 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 7.92 (d,J=8 Hz, 1H), 5.82 (d, J=5.2 Hz, 1H), 5.68-5.63 (m, 1H), 5.20-5.15 (m,1H), 4.32-4.25 (m, 1H), 3.87-3.80 (m, 2H), 3.69-3.61 (m, 1H), 3.57-3.49(m, 1H), 0.88 (s, 9H), 0.09 (s, 6H).

Preparation of (6): To the solution of 5 (6.5 g, 17.2 mmol) in dry DCM(35 mL) and DMF (9 mL) was added PDC (12.9 g, 34.3 mmol), tert-butylalcohol (34 mL) and Ac₂O (17 mL) at r.t under N₂ atmosphere. And thereaction mixture was stirred at r.t for 2 hrs. The solvent was removedto give a residue which was purified by silica gel column chromatography(eluent, PE: EA=4:1-2:1) to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give 6 (5.5 g, 12.3 mmol, 70.1%) as a white solid.ESI-LCMS: m/z 446 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ=11.29 (s, 1H),7.91 (d, J=8.4 Hz, 1H), 5.85 (d, J=6.4 Hz, 1H), 5.71-5.61 (m, 1H),4.35-4.28 (m, 1H), 4.12 (d, J=3.2 Hz, 1H), 3.75-3.67 (m, 1H), 1.33 (s,9H), 0.76 (s, 9H), 0.00 (d, J=1.6 Hz, 6H).

Preparation of (7): To the solution of 6 (5.4 g, 12.1 mmol) inTHF/MeOD/D20=10/2/1 (44 mL) was added NaBD₄ (1.5 g, 36.3 mmol) at r.t.and the reaction mixture was stirred at 50° C. for 2 hrs. Aftercompletion of reaction, adjusted pH value to 7 with CH₃COOD. Water wasadded, the resulting mixture was extracted with EA (500 mL). Thecombined organic layer was washed with water and brine, dried overNa₂SO₄, and concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 7 (2.6 g, 6.8 mmol, 56.1%) as a white solid.ESI-LCMS: m/z 378 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.35 (s, 1H),7.91 (d, J=8.0 Hz, 1H), 5.82 (d, J=5.2 Hz, 1H), 5.69-5.60 (m, 1H), 5.14(s, 1H), 4.34-4.20 (m, 1H), 3.88-3.76 (m, 2H), 0.87 (s, 9H), 0.08 (s,6H).

Preparation of (8): To a stirred solution of 7 (2.6 g, 6.8 mmol) inpyridine (30 mL) were added DMTrCl (3.5 g, 10.3 mmol) at r.t. And thereaction mixture was stirred at r.t. for 2.5 hrs. With ice-bath cooling,the reaction was quenched with water and the product was extracted intoEA (200 mL). The organic phase was evaporated to dryness under reducedpressure to give a residue which was purified by Flash-Prep-HPLC withthe following conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 20 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in togive 8 (4.3 g, 6.3 mmol, 90.1%) as a white solid. ESI-LCMS: m/z 678[M−H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ 11.39 (s, 1H), 7.86 (d, J=8.0 Hz,1H), 7.42-7.17 (m, 9H), 6.96-6.83 (m, 4H), 5.82-5.69 (m, 2H), 5.29 (d,J=8.4 Hz, 1H), 4.36-4.25 (m, 1H), 3.90 (d, J=7.2 Hz, 1H), 3.86-3.80 (m,1H), 3.73 (s, 6H), 0.75 (s, 9H), 0.02 (s, 3H), −0.04 (s, 3H).

Preparation of (9): To a solution of 8 (4.3 g, 6.3 mmol) in THF (45 mL)was added 1 M TBAF solution (6 mL). The reaction mixture was stirred atr.t. for 1.5 hrs. LCMS showed 8 was consumed completely. Water (200 mL)was added. The product was extracted with EA (200 mL) and the organiclayer was washed with brine and dried over Na₂SO₄. Then the organiclayer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 8 (3.5 g, 6.1 mmol, 90.1%) as a white solid.ESI-LCMS: m/z 678 [M−H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ 11.38 (d, J=2.0Hz, 1H), 7.23 (d, J=8.0 Hz, 1H), 7.41-7.19 (m, 9H), 6.94-6.85 (m, 4H),5.81 (d, J=4.0 Hz, 1H), 5.33-5.26 (m, 1H), 5.21 (d, J=7.2 Hz, 1H),4.06-3.90 (m, 2H), 3.83-3.77 (m, 1H), 3.74 (s, 6H).

Preparation of Example 25 monomer: To a suspension of 9 (2.1 g, 3.7mmol) in DCM (20 mL) was added DCI (373 mg, 3.1 mmol) and CEP[N(iPr)₂]₂(1.3 g, 4.4 mmol). The mixture was stirred at r.t. for 1 h. LC-MS showed9 was consumed completely. The solution was washed with water twice andwashed with brine and dried over Na₂SO₄. Then concentrated to give aresidue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 25monomer (2.2 g, 3.5 mmol, 80%) as a white solid. ESI-LCMS: m/z 766[M+H]⁺; ¹H-NMR (400 MHz, ACN-d3): S 9.65-8.86 (m, 1H, exchanged withD20), 7.93-7. 68 (m, 1H), 7.52-7.19 (m, 9H), 6.94-6.78 (m, 4H),5.95-5.77 (m, 1H), 5.31-5.17 (m, 1H), 4.61-4.37 (m, 1H), 4.20-4.07 (m,1H), 4.01-3.51 (m, 10H), 2.74-2.59 (m, 1H), 2.57-2.43 (m, 1H), 1.27-1.10(m, 9H), 1.09-0.95 (m, 3H). ³¹P-NMR (162 MHz, ACN-d3): δ=149.88, 149.55.

Example 26. Synthesis of Monomer

Preparation of (7): To a solution of 6 (17 g, 25.1 mmol, Scheme 3) inACN (170 mL) was added DMAP (6.13 g, 50.3 mmol) and TEA (5.1 g, 50.3mmol, 7.2 mL), Then added TPSCI (11.4 g, 37.7 mmol) at 0° C. under N₂atmosphere and the mixture was stirred at r.t. for 3 h under N₂atmosphere. Then con. NH₃.H₂O (27.3 g, 233.7 mmol) was added at r.t. andthe mixture was stirred at r.t. for 16 h. The reaction was quenched withwater and the product was extracted with EA (200 mL). The organic phasewas concentrated to give the crude 7 (17.0 g) as a white solid which wasused directly for next step.

Preparation of (8): To a stirred solution of 7 (17.0 g, 25.1 mmol) inpyridine (170 mL) were added BzCl (4.3 g, 30.1 mmol) 0° C. under N₂atmosphere. And the reaction mixture was stirred at r.t for 2.5 h. Withice-bath cooling, the reaction was quenched with water and the productwas extracted with EA (200 mL). The organic phase was evaporated todryness under reduced pressure to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give 8 (19.0 g, 24.3 mmol, 95.6% over two step) as awhite solid. ESI-LCMS: m/z 780 [M+H]⁺.

Preparation of (9): To a solution of 8 (19.0 g, 24.3 mmol) in THF (190mL) was added 1 M TBAF solution (24 mL). The reaction mixture wasstirred at r.t. for 1.0 h. LC-MS showed 8 was consumed completely. Water(500 mL) was added. The product was extracted with EA (300 mL) and theorganic layer was washed with brine and dried over Na₂SO₄. Then theorganic layer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 9 (15.2 g, 23.1 mmol, 95.5%) as a white solid.ESI-LCMS: m/z 666 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.28 (s, 1H),8.41 (m, 1H), 8.00-7.99 (m, 2H), 7.63-7.15 (m, 13H), 6.93-6.89 (m, 4H),5.87 (s, 1H), 5.20 (d, J=7.4 Hz, 1H), 4.30 (m, 1H), 4.02 (m, 1H), 3.75(s, 7H), 3.53 (s, 3H).

Preparation of Example 26 monomer: To a suspension of 9 (10.0 g, 15.0mmol) in DCM (100 mL) was added DCI (1.5 g, 12.7 mmol) and CEP[N(iPr)₂]₂(5.4 g, 18.0 mmol). The mixture was stirred at r.t. for 1 h. LC-MSshowed 9 was consumed completely. The solution was washed with watertwice and washed with brine and dried over Na₂SO₄. Then concentrated togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 26monomer (11.5 g, 13.5 mmol, 90.7%) as a white solid. ESI-LCMS: m/z 866[M+H]+, ¹H-NMR (400 MHz, DMSO-d6): δ=11.28 (s, 1H), 8.48-8.41 (m, 1H),8.00-7.99 (m, 2H), 7.63-7.11 (m, 13H), 6.93-6.89 (m, 4H), 5.92 (m, 1H),4.55-4.44 (m, 1H), 4.17 (m, 1H), 3.95 (m, 1H), 3.80-3.62 (m, 7H),3.57-3.46 (m, 5H), 3.32 (s, 1H), 2.78 (m, 1H), 2.62-2.59 (m, 1H),1.19-0.94 (m, 12H); ³¹P-NMR (162 MHz, DMSO-d6): δ=149.52, 148.82.

Example 27. Synthesis of Monomer

Preparation of (5): To the solution of 4 (18.8 g, Scheme 5) in dry ACN(200 mL) was added TPSCI (16.8 g, 65.2 mmol) and TEA (5.6 g, 65.2 mmol)and DMAP (6.8 g, 65.2 mmol), and the reaction mixture was stirred atroom temperature for 3.5 hrs under N₂ atmosphere. After addition ofwater, the resulting mixture was extracted with EA (300 mL). Thecombined organic layer was washed with water and brine, dried overNa₂SO₄, and concentrated to give the crude 5 (22.0 g) as a white solidwhich was used directly for next step. ESI-LCMS: m/z 677 [M−H]⁺.

Preparation of (6): To a solution of 5 (22.0 g) in pyridine (150 mL) wasadded BzCl (6.8 g, 48.9 mmol) under ice bath. The reaction mixture wasstirred at r.t. for 2.5 hrs. LCMS showed 5 was consumed. The mixture wasdiluted with EA and water was added. The product was extracted with EA.The crude was purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give the crude 6 (20.8 g, 26.7mmol, 82% yield over two steps) as a white solid. ESI-LCMS: m/z 781[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.30 (s, 1H), 8.55 (d, J=8.0 Hz,1H), 8.00-7.98 (m, 2H), 7.74-7.66 (m, 1H), 7.60-7.50 (m, 2H), 7.47-7.31(m, 4H), 7.30-7.2(m, 5H), 7.20-7.1(m, 1H), 6.91 (d, J=7.4 Hz, 4H),5.91-5.86 (AB, J=20.0 Hz, 1H), 4.30 (d, J=8.0 Hz, 1H), 3.87-3.78 (s,1H), 3.78-3.70 (m, 6H), 3.62-3.51 (m, 1H), 3.28-3.2 (m, 1H), 2.15-2.05(m, 3H), 0.73 (s, 9H), 0.00 (m, 6H).

Preparation of (7): To a solution of 6 (20.8 g, 26.7 mmol) in THF (210mL) was added 1 M TBAF solution (32 mL). The reaction mixture wasstirred at r.t. for 1.5 hrs. LCMS showed 6 was consumed completely.Water (600 mL) was added. The product was extracted with EA (400 mL) andthe organic layer was washed with brine and dried over Na₂SO₄. Then theorganic layer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 7 (12.4 g, 18.6 mmol, 70%) as a white solid.ESI-LCMS: m/z 667 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.03 (m, 1H),8.51-8.48 (m, 1H), 8.08-7.95 (m, 2H), 7.63-7.54 (m, 1H), 7.52-7.19 (m,9H), 7.16-7.07 (m, 1H), 6.94-6.89 (m, 3H), 5.95-5.87 (m, 1H), 5.31-5.17(m, 1H), 4.61-4.37 (m, 1H), 4.20-4.07 (m, 1H), 3.82-3.47 (m, 7H),2.57-2.42 (m, 2H).

Preparation of Example 27 monomer: To a suspension of 7 (12.4 g, 18.6mmol) in DCM (120 mL) was added DCI (1.7 g, 15.8 mmol) and CEP[N(iPr)₂]₂(7.3 g, 24.2 mmol). The mixture was stirred at r.t. for 2 hrs. LC-MSshowed 7 was consumed completely. The solution was washed with watertwice and washed with brine and dried over Na₂SO₄. Then concentrated togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 27monomer (13.6 g, 15.7 mmol, 84.0%) as a white solid. ESI-LCMS: m/z 867[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.03 (m, 1H), 8.51-8.48 (m, 1H),8.08-7.95 (m, 2H), 7.63-7.54 (m, 1H), 7.52-7.19 (m, 9H), 7.16-7.07 (m,1H), 6.94-6.89 (m, 3H), 5.95-5.87 (m, 1H), 5.31-5.17 (m, 1H), 4.61-4.37(m, 1H), 4.20-4.07 (m, 1H), 3.82-3.47 (m, 10H), 2.74-2.59 (m, 1H),2.57-2.43 (m, 1H), 1.27-1.10 (m, 9H), 1.09-0.95 (m, 3H). ³¹P-NMR (162MHz, DMSO-d6): δ 149.59, 148.85.

Example 28. Synthesis of Monomer

Preparation of (4): To a solution of 3 (13.1 g, 35.2 mmol, Scheme 3) inpyridine (130 mL) was added MsCl (4.8 g, 42.2 mmol) under −10 ˜0° C. Thereaction mixture was stirred at r.t. for 2.5 h under N₂ atmosphere. TLC(DCM/MeOH=15:1) showed the reaction was consumed. The mixture wasdiluted with EA and water was added. The product was extracted with EA.The organic layer was washed with brine and dried over Na₂SO₄ andconcentrated to give the crude. This resulted in to give the product 4(14.2 g) which was used directly for the next step. ESI-LCMS: m/z 451[M+H]⁺; H-NMR (400 MHz, DMSO-d6) δ 11.43 (m, 1H), 7.67-7.65 (m, 1H),5.90-5.80 (m, 1H), 5.75-5.64 (m, 1H), 4.52-4.21 (m, 3H), 4.12-3.90 (m,2H), 3.48-3.21 (m, 6H), 0.95-0.78 (s, 9H), 0.13-0.03 (s, 6H).

Preparation of (5): To a solution of 4 (14.2 g) in DMSO (200 mL) wasadded DMTrSH (19.6 g, 63.2 mmol) and tetramethylguanidine (5.1 g, 47.4mmol) at r.t. The reaction mixture was stirred at r.t. for 3.5 h underN₂ atmosphere. LCMS showed 4 the reaction was consumed. The mixture wasdiluted with EA and water was added. The product was extracted with EA.The organic layer was washed with brine and dried over Na₂SO₄ andconcentrated to give the crude. The crude was purified by silica gelcolumn (SiO₂, PE/EA=10:1-1:1) to give 5 (14.2 g, 20.6 mmol, 58.5% yieldover two steps) as a white solid. ESI-LCMS: m/z 689 [M+H]; ¹H-NMR (400MHz, DMSO-d6) δ 11.39 (m, 1H), 7.63-7.61 (d, J=8.0 Hz, 1H), 7.45-7.1(m,9H), 6.91-6.81 (m, 4H), 5.80-5.70 (m, 2H), 4.01-3.91 (m, 1H), 3.85-3.78(m, 1H), 3.78-3.65 (m, 6H), 3.60-3.51 (m, 1H), 3.43-3.2(m, 3H),2.50-2.32 (m, 2H), 0.95-0.77 (s, 9H), −0.00-0.02 (s, 6H).

Preparation of (6): To a solution of 5 (14.2 g, 20.6 mmol) in THF (140mL) was added 1 M TBAF solution (20 mL). The reaction mixture wasstirred at r.t. under N₂ atmosphere for 2.5 h. LCMS showed 5 wasconsumed completely. Water was added. The product was extracted with EAand the organic layer was washed with brine and dried over Na₂SO₄. Thenthe organic layer was concentrated to give a residue which was purifiedby Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 6 (10.5 g, 18.2 mmol, 88.5%) as a white solid.ESI-LCMS: m/z 576 [M+H]⁺; H-NMR (400 MHz, DMSO-d6) δ 11.38 (m, 1H),7.56-7.54 (d, J=8.0 Hz, 1H), 7.45-7.1(m, 9H), 6.91-6.81 (m, 4H),5.80-5.70 (m, 2H), 4.05-4.00 (m, 1H), 3.81-3.79 (m, 1H), 3.74 (m, 2H),3.78-3.65 (m, 6H), 3.60-3.51 (m, 1H), 3.43-3.2(m, 3H), 2.40-2.32 (m,1H).

Preparation of Example 28 monomer: To a suspension of 9 (10.5 g, 18.2mmol) in DCM (100 mL) was added DCI (1.7 g, 15.5 mmol) and CEP[N(iPr)₂]₂(7.2 g, 23.7 mmol). The mixture was stirred at r.t. for 1 h. LC-MSshowed 9 was consumed completely. The solution was washed with watertwice and washed with brine and dried over Na₂SO₄. Then concentrated togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 28monomer (12.5 g, 16.1 mmol, 88%) as a white solid. ESI-LCMS: m/z 776[M+H]⁺ H-NMR (400 MHz, DMSO-d6) δ 11.41 (m, 1H), 7.64-7.59 (m, 1H),7.40-7.25 (m, 4H), 7.25-7.10 (m, 5H), 6.89-6.86 (m, 4H), 5.72-5.67 (m,2H), 4.02-4.00 (m, 2H), 3.76-3.74 (m, 8H), 3.74-3.73 (m, 3H), 3.51-3.49(d, J=8 Hz, 1H), 3.33-3.29 (m, 1H), 2.77-2.73 (m, 1H), 2.63-2.60 (m,1H), 2.50-2.47 (m, 1H), 1.12-0.99 (m, 12H). ³¹P-NMR (162 MHz, DMSO-d6):δ 148.92, 148.84.

Example 29. Synthesis of Monomer

Preparation of (7): To a solution of 6 (16 g, 24.1 mmol, Scheme 4) inACN (160 mL) was added DMAP (5.9 g, 48.2 mmol) and TEA (4.8 g, 48.2mmol), then added TPSCI (10.9 g, 36.1 mmol) at 0° C. under N₂ atmosphereand the mixture was stirred at r.t. for 5 hrs under N₂ atmosphere. Thencon. NH₃.H₂O (30 mL) was added at r.t. and the mixture was stirred atr.t. for 16 h. The reaction was quenched with water and the product wasextracted with EA (200 mL). The organic phase was concentrated to givethe crude 7 (16.0 g) as a white solid which was used directly for nextstep.

Preparation of (8): To a stirred solution of 7 (16.0 g, 24.1 mmol) inpyridine (160 mL) were added BzCl (4.1 g, 28.9 mmol) 0° C. under N₂atmosphere. And the reaction mixture was stirred at r.t. for 2.5 h. Withice-bath cooling, the reaction was quenched with water and the productwas extracted with EA (200 mL). The organic phase was evaporated todryness under reduced pressure to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give 8 (18.0 g, 23.4 mmol, 97.0%) as a white solid.ESI-LCMS: m/z 768 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.31 (s, 1H),8.47 (d, J=7.2 Hz, 1H), 7.99 (d, J=7.6 Hz, 2H), 7.65-7.16 (m, 13H), 6.92(d, J=8.8 Hz, 4H), 6.01 (d, J=18.4 Hz, 1H), 5.18-5.04 (dd, 1H),4.58-4.52 (m, 1H), 4.07 (d, J=9.6 Hz, 1H), 3.75 (s, 6H), 0.73 (s, 9H),0.05 (s, 3H), −0.06 (s, 3H).

Preparation of (9): To a solution of 8 (18.0 g, 23.4 mmol) in THF (180mL) was added 1 M TBAF solution (23 mL). The reaction mixture wasstirred at r.t. for 1.5 h. LC-MS showed 8 was consumed completely. Water(500 mL) was added. The product was extracted with EA (300 mL) and theorganic layer was washed with brine and dried over Na₂SO₄. Then theorganic layer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 7 (13.7 g, 21.1 mmol, 90.5%) as a white solid.ESI-LCMS: m/z 654.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.31 (s, 1H),8.35 (d, J=7.4 Hz, 1H), 8.01 (m, 2H), 7.65-7.16 (m, 13H), 6.92 (d, J=8.8Hz, 4H), 5.94 (d, J=18.0 Hz, 1H), 5.71 (d, J=7.0 Hz, 1H), 5.12-4.98 (dd,1H), 4.51-4.36 (m, 1H), 4.09 (d, J=9.6 Hz, 1H), 3.75 (s, 6H).

Preparation of Example 29 monomer: To a suspension of 9 (10.6 g, 16.2mmol) in DCM (100 mL) was added DCI (1.6 g, 13.7 mmol) and CEP[N(iPr)₂]₂(5.8 g, 19.4 mmol). The mixture was stirred at r.t. for 1 h. LC-MSshowed 9 was consumed completely. The solution was washed with watertwice and washed with brine and dried over Na₂SO₄. Then concentrated togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 29monomer (10.5 g, 14.5 mmol, 75.9%) as a white solid. ESI-LCMS: m/z 854.3[M+H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ 11.31 (s, 1H), 8.41-8.37 (m, 1H),8.01 (d, J=7.7 Hz, 2H), 7.65-7.16 (m, 13H), 6.92-6.88 (m, 4H), 6.06-5.98(m, 1H), 5.33-5.15 (m, 1H), 4.78-4.58 (m, 1H), 4.23-4.19 (m, 1H),3.81-3.73 (m, 6H), 3.60-3.50 (m, 3H), 3.32 (s, 1H), 2.76 (t, J=6.0 Hz,1H), 2.60 (t, J=5.8 Hz, 1H), 1.15-0.94 (m, 12H); ³¹P-NMR (162 MHz,DMSO-d6): δ 150.23, 150.18, 149.43, 149.38.

Example 30. Synthesis of Monomer

Preparation of (9): To a solution of 8 (18.8 g, 26.4 mmol, Scheme 5) inACN (200 mL) was added TPSCI (16.8 g, 55.3 mmol) and DMAP (5.6 g, 55.3mmol) and TEA (6.8 g, 55.3 mmol). The reaction mixture was stirred atr.t. for 3.5 hrs. LCMS showed the reaction was consumed. The mixture wasdiluted with con. NH₄OH (28 mL). The mixture was diluted with water andEA. The product was extracted with EA. The organic layer was washed withbrine and dried over Na₂SO₄ and concentrated to give the crude 9 (18.5g) which was used directly for the next step.

Preparation of (10): To a solution of 9 (18.8 g, 27.69 mmol) in pyridine(200 mL) was added BzCl (5.8 g, 41.5 mmol) under ice bath. The reactionmixture was stirred at r.t. for 2.5 hrs. LCMS showed 9 was consumed. Themixture was diluted with EA and water was added. The product wasextracted with EA. The crude was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 25 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in togive 10 (19.8 g, 25.3 mmol, 91% yield) as a white solid. ESI-LCMS: m/z783 [M−H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ 11.29 (d, J=2.0 Hz, 1H), 8.42(d, J=8.0 Hz, 1H), 8.02-8.00 (m, 2H), 7.64-7.62 (m, 1H), 7.60-7.41 (m,2H), 7.47.41-7.19 (m, 9H), 6.94-6.85 (m, 4H), 5.81 (d, J=4.0 Hz, 1H),5.33-5.26 (m, 1H), 5.21 (d, J=7.2 Hz, 1H), 4.06-3.90 (m, 2H), 3.83-3.77(m, 1H), 3.74 (s, 6H).

Preparation of (11): To a solution of 10 (18.8 g, 26.4 mmol) in THF (190mL) was added 1 M TBAF solution (28 mL). The reaction mixture wasstirred at r.t. for 1.5 hrs. LCMS showed 10 was consumed completely.Water (200 mL) was added. The product was extracted with EA (200 mL) andthe organic layer was washed with brine and dried over Na₂SO₄. Then theorganic layer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 11 (17.1 g, 25.6 mmol, 96%) as a white solid.ESI-LCMS: m/z 669 [M−H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ 11.29 (d, J=2.0Hz, 1H), 8.42 (d, J=8.0 Hz, 1H), 8.02-8.00 (m, 2H), 7.64-7.62 (m, 1H),7.60-7.41 (m, 2H), 7.47.41-7.19 (m, 9H), 6.94-6.85 (m, 4H), 5.81 (d,J=4.0 Hz, 1H), 5.33-5.26 (m, 1H), 5.21 (d, J=7.2 Hz, 1H), 4.06-3.90 (m,2H), 3.83-3.77 (m, 1H), 3.74 (s, 6H).

Preparation of Example 30 monomer: To a suspension of 11(10.8 g, 16.2mmol) in DCM (100 mL) was added DCI (1.5 g, 13.7 mmol) and CEP[N(iPr)₂]₂(5.8 g, 19.3 mmol). The mixture was stirred at r.t. for 2 hrs. LC-MSshowed 11 was consumed completely. The solution was washed with watertwice and washed with brine and dried over Na₂SO₄. Then concentrated togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 30monomer (11.3 g, 13 mmol, 80%) as a white solid. ESI-LCMS: m/z 868[M+H]; ¹H-NMR (400 MHz, DMSO-d6): δ 11.03 (m, 1H), 8.51-8.48 (m, 1H),8.08-7.95 (m, 2H), 7.63-7.54 (m, 1H), 7.52-7.19 (m, 9H), 7.16-7.07 (m,1H), 6.94-6.89 (m, 3H), 5.95-5.87 (m, 1H), 5.31-5.17 (m, 1H), 4.61-4.37(m, 1H), 4.20-4.07 (m, 1H), 3.82-3.47 (m, 10H), 2.74-2.59 (m, 1H),2.57-2.43 (m, 1H), 1.27-1.10 (m, 9H), 1.09-0.95 (m, 3H). ³¹P-NMR (162MHz, DMSO-d6): δ 149.52, 148.81.

Example 31. Synthesis of Monomer

Preparation of (2): To a stirred solution of 1 (100.0 g, 406.5 mmol) inpyridine (1000 mL) were added DMTrCl (151.2 g, 447.1 mmol) at r.t. Andthe reaction mixture was stirred at r.t. for 2.5 hrs. With ice-bathcooling, the reaction was quenched with water and the product wasextracted with EA (3000 mL). The organic phase was evaporated to drynessunder reduced pressure to give a residue which was purified by silicagel column chromatography (SiO₂, dichloromethane: methanol=100:1) togive 2 (210.0 g, 90%) as a white solid. ESI-LCMS: m/z 548.2 [M+H]⁺;¹H-NMR (400 MHz, DMSO-d6): δ 11.43 (d, J=1.8 Hz, 1H), 7.77 (d, J=8.0 Hz,1H), 7.40-7.21 (m, 9H), 6.92-6.88 (m, 4H), 5.89 (d, J=20.0 Hz, 1H),5.31-5.29 (m, 1H), 5.19-5.04 (dd, 1H), 4.38-4.31 (m, 1H), 4.02-3.98 (m,1H), 3.74 (s, 6H), 3.30 (d, J=3.2 Hz, 2H); ¹⁹F-NMR (376 MHz, DMSO-d6): δ−199.51.

Preparation of (3): To a stirred solution of 2 (100.0 g, 182.8 mmol) inpyridine (1000 mL) were added MsCl (31.2 g, 274.2 mmol) at 0° C. underN₂ atmosphere. And the reaction mixture was stirred at r.t for 2.5 h.With ice-bath cooling, the reaction was quenched with water and theproduct was extracted with EA (200 mL). The organic phase was evaporatedto dryness under reduced pressure to give the crude (114.0 g) as a whitesolid which was used directly for next step. To the solution of thecrude (114.0 g, 187.8 mmol) in DMF (2000 mL) was added K₂CO₃ (71.5 g,548.4 mmol), and the reaction mixture was stirred at 90° C. for 15 hunder N₂ atmosphere. After addition of water, the resulting mixture wasextracted with EA (500 mL). The combined organic layer was washed withwater and brine, dried over Na₂SO₄, and concentrated to give a residuewhich was purified by silica gel column chromatography (SiO₂,dichloromethane: methanol=30:1) to give 3 (100.0 g, 90%) as a whitesolid. ESI-LCMS: m/z 531.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 7.79 (d,J=8.0 Hz, 1H), 7.40-7.21 (m, 9H), 6.89-6.83 (m, 4H), 6.14 (d, J=5.4 Hz,1H), 6.02-5.90 (dd, 1H), 5.87 (d, J=20.0 Hz, 1H), 5.45 (m, 1H), 4.61 (m,1H), 3.73 (d, J=1.9 Hz, 6H), 3.30-3.15 (m, 2H), 1.24-1.16 (m, 1H);¹⁹F-NMR (376 MHz, DMSO-d6): δ −204.23.

Preparation of (4): A solution of 3 (100 g, 187.8 mmol) in THF (1000 mL)was added 6N NaOH (34 mL, 206.5 mmol). The mixture was stirred at r.t.for 6 h. After completion of reaction, the resulting mixture was addedH₂O, and then the mixture was extracted with EA, the organic layer waswashed with brine, dried over sodium sulfate and removed to give theresidue was purified by silica gel column chromatography (SiO₂,dichloromethane: methanol=30:1) to give 4 (90.4 g, 90%) as a whitesolid. ESI-LCMS: m/z 548.2 [M+H]⁺; ¹⁹F-NMR (376 MHz, DMSO-d6): δ−184.58.

Preparation of (5): To a stirred solution of 4 (90.4 g, 165.2 mmol) inpyridine (1000 mL) were added MsCl (61.5 g, 495.6 mmol) at 0° C. underN₂ atmosphere. And the reaction mixture was stirred at r.t for 16 hrs.With ice-bath cooling, the reaction was quenched with water and theproduct was extracted with EA. the organic layer was washed with brine,dried over sodium sulfate and removed to give the residue was purifiedby silica gel column chromatography (SiO₂, PE: EA=1:1) to give 5 (75.0g, 90%) as a white solid. ESI-LCMS: m/z 626.2 [M+H]⁺; ¹H-NMR (400 MHz,DMSO-d6): δ 11.51 (d, J=1.6 Hz, 1H), 7.43-7.23(m, 10H), 6.92-6.88 (m,4H), 6.08 (d, J=20.0 Hz, 1H), 5.55-5.39 (m, 2H), 4.59 (m, 1H), 3.74 (s,6H), 3.48-3.28 (m, 2H), 3.17 (s, 3H); ¹⁹F-NMR (376 MHz, DMSO-d6): δ−187.72.

Preparation of (6): To the solution of 5 (75.0 g, 120.4 mmol) in DMF(1500 mL) was added KSAc (71.5 g, 548.4 mmol) at 110° C. under N₂atmosphere, After the reaction mixture was stirred at 110° C. for 3 hwere added KSAc (71.5 g, 548.4 mmol) under N₂ atmosphere. And thereaction mixture was stirred at r.t for 16 h. After addition of water,the resulting mixture was extracted with EA. The combined organic layerwas washed with water and brine, dried over Na₂SO₄, and concentrated togive a residue which was purified by silica gel column chromatography(SiO₂, PE: EA=1:1) to give 6 (29.0 g, 90%) as a white solid. ESI-LCMS:m/z 605.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.45 (d, J=1.9 Hz, 1H),7.95 (d, J=8.0 Hz, 1H), 7.38-7.21 (m, 9H), 6.92-6.87 (m, 4H), 5.93 (m,1H), 5.50-5.36 (dd, 1H), 5.25-5.23 (dd, 1H), 4.54-4.42 (m, 1H),4.17-4.12 (m, 1H), 3.74 (m, 7H), 3.35-3.22 (m, 2H), 2.39 (s, 1H);¹⁹F-NMR (376 MHz, DMSO-d6): δ −181.97.

Preparation of (7): A solution of 6 (22 g, 36.3 mmol) in a mixturesolvent of THF/MeOH (1:1, 200 mL) was added 1N NaOMe (70 mL, 72.6mmol)was stirred at 20° C. for 4 h. After completion of reaction, theresulting mixture was added H₂O, and then the mixture was extracted withEA, the organic layer was washed with brine, dried over sodium sulfateand removed to give the residue was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=3/2 within 25 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=4/3; Detector, UV 254 nm. This resulted in togive 7 (10.5 g, 14.5 mmol, 75.9%) as a white solid. ESI-LCMS: m/z 565.1[M+H]⁺: ¹H-NMR (400 MHz, DMSO-d6): δ 11.45 (s, 1H), 7.83 (d, J=8.0 Hz,1H), 7.40-7.23 (m, 9H), 6.90 (d, J=8.8 Hz, 4H), 5.88 (m, 1H), 5.29-5.15(m, 2H), 3.72 (m, 7H), 3.43 (m, 2H), 2.78 (d, J=10.6 Hz, 1H).

Preparation of Example 31 monomer: To a suspension of 7 (10.5 g, 18.6mmol) in DCM (100 mL) was added DCI (1.8 g, 15.7 mmol) and CEP[N(iPr)₂]₂(6.7 g, 22.3 mmol). The mixture was stirred at r.t. for 1 h. LC-MSshowed 8 was consumed completely. The solution was washed with watertwice and washed with brine and dried over Na₂SO₄. Then concentrated togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 31monomer (10.5 g, 14.5 mmol, 75.9%) as a white solid. ESI-LCMS: m/z 765.3[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.40 (d, J=12.2 Hz, 1H), 7.90-7.86(m, 1H), 7.41-7.24 (m, 9H), 6.91-6.89 (m, 4H), 5.97 (m, 1H), 5.33-5.10(m, 2H), 4.18-4.16 (m, 1H), 3.91-3.39 (m, 17H), 2.81 (t, J=5.6 Hz, 1H),2.66 (t, J=6.0 Hz, 1H), 1.33-0.97 (m, 12H); ³¹P-NMR (162 MHz, DMSO-d6):δ 164.57, 160.13.

Example 32. Synthesis of Monomer

Preparation of (2): To a stirred solution of 1 (100.0 g, 387.5 mmol) inpyridine (1000 mL) was added DMTrCl (151.2 g, 447.1 mmol) at r.t. Andthe reaction mixture was stirred at r.t. for 2.5 hrs. With ice-bathcooling, the reaction was quenched with water and the product wasextracted with EA (3000 mL). The organic phase was evaporated to drynessunder reduced pressure to give a residue which was purified by silicagel column chromatography (SiO₂, dichloromethane: methanol=100:1) togive 2 (200.0 g, 90%) as a white solid. ESI-LCMS: m/z 561 [M+H]⁺.

Preparation of (3): To a stirred solution of 2 (73.0 g, 130.3 mmol) inpyridine (730 mL) were added MsCl (19.5 g, 169.2 mmol) at 0° C. under N₂atmosphere. And the reaction mixture was stirred at r.t for 2.5 h. Withice-bath cooling, the reaction was quenched with water and the productwas extracted with EA (200 mL). The organic phase was evaporated todryness under reduced pressure to give the crude (80.0 g) as a whitesolid which was used directly for next step. To the solution of thecrude (80.0 g, 130.3 mmol) in DMF (1600 mL) was added K₂CO₃ (71.5 g,390.9 mmol), and the reaction mixture was stirred at 90° C. for 15 hunder N₂ atmosphere. After addition of water, the resulting mixture wasextracted with EA (500 mL). The combined organic layer was washed withwater and brine, dried over Na₂SO₄, and concentrated to give a residuewhich was purified by silica gel column chromatography (SiO₂,dichloromethane: methanol=30:1) to give 3 (55.0 g, 90%) as a whitesolid. ESI-LCMS: m/z 543. [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 7.68 (d,J=8.0 Hz, 1H), 7.40-7.21 (m, 9H), 6.89-6.83 (m, 4H), 5.96 (s, 1H), 5.83(d, J=5.4 Hz, 1H), 5.26 (s, 1H), 4.59 (s, 1H), 4.46 (t, J=6.0 Hz, 1H),3.72 (s, 6H), 3.44 (s, 3H), 3.18-3.12 (m, 2H).

Preparation of (4): A solution of 3 (55 g, 101.8 mmol) in THF (550 mL)was added 6N NaOH (34 mL, 206.5 mmol). The mixture was stirred at 20° C.for 6 hrs. After completion of reaction, the resulting mixture was addedH₂O, and then the mixture was extracted with EA, the organic layer waswashed with brine, dried over sodium sulfate and removed to give theresidue was purified by silica gel column chromatography (SiO₂,dichloromethane: methanol=30:1) to give 4 (57.4 g, 87%) as a whitesolid. ESI-LCMS: m/z 561 [M+H]⁺.

Preparation of (5): To a stirred solution of 4 (57.4 g, 101.8 mmol) inpyridine (550 mL) were added MsCl (61.5 g, 495.6 mmol) at 0° C. under N₂atmosphere. And the reaction mixture was stirred at r.t for 16 h. Withice-bath cooling, the reaction was quenched with water and the productwas extracted with EA. the organic layer was washed with brine, driedover sodium sulfate and removed to give the residue was purified bysilica gel column chromatography (SiO₂, PE: EA=1:1) to give 5 (57.0 g,90%) as a white solid. ESI-LCMS: m/z 639 [M+H]⁺.

Preparation of (6): To the solution of 5 (57.0 g, 89.2 mmol) in DMF (600mL) was added KSAc (71.5 g, 448.4 mmol) at 110° C. under N₂ atmosphere,After the reaction mixture was stirred at 110° C. for 3 h were addedKSAc (71.5 g, 448.4 mmol) under N₂ atmosphere. And the reaction mixturewas stirred at r.t for 16 h. After addition of water, the resultingmixture was extracted with EA. The combined organic layer was washedwith water and brine, dried over Na₂SO₄, and concentrated to give aresidue which was purified by silica gel column chromatography (SiO₂,PE: EA=1:1) to give 6 (29.0 g, 47%) as a white solid. ESI-LCMS: m/z619.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.41 (s, 1H), 8.06 (s, 1H),7.40-7.23 (m, 9H), 6.90 (d, J=8.8 Hz, 4H), 5.82 (s, 1H), 5.10-5.08 (dd,1H), 4.38-4.34 (m, 1H), 4.08-4.02 (m, 3H), 3.74 (s, 6H), 3.45 (s, 3H),3.25 (m, 2H), 2.37 (s, 3H); ESI-LCMS: m/z 619 [M+H]⁺.

Preparation of (7): A solution of 6 (22 g, 35.3 mmol) in a mixturesolvent of THF/MeOH (1:1, 200 mL) was added 1N NaOMe (70 mL, 72.6mmol)was stirred at 20° C. for 4 h. After completion of reaction, theresulting mixture was added H₂O, and then the mixture was extracted withEA, the organic layer was washed with brine, dried over sodium sulfateand removed to give the residue was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=3/2 within 25 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=4/3; Detector, UV 254 nm. This resulted in togive 7 (14.0 g, 70.9%) as a white solid. ESI-LCMS: m/z 576.1 [M+H]⁺;¹H-NMR (400 MHz, DMSO-d6): δ 11.38 (s, 1H), 7.90 (d, J=8.0 Hz, 1H),7.40-7.23 (m, 9H), 6.90 (d, J=8.8 Hz, 4H), 5.80 (s, 1H), 5.15-5.13 (dd,1H), 3.93 (m, 1H), 3.87 (d, J=5.0 Hz, 1H), 3.74 (s, 6H), 3.59 (m, 2H),3.49 (s, 3H), 3.39 (d, J=2.2 Hz, 2H), 2.40 (d, J=10.2 Hz, 1H).

Preparation of Example 32 monomer: To a suspension of 7 (10.5 g, 18.6mmol) in DCM (100 mL) was added DCI (1.8 g, 15.7 mmol) and CEP[N(iPr)₂]₂(6.7 g, 22.3 mmol). The mixture was stirred at r.t. for 1 h. LC-MSshowed 7 was consumed completely. The solution was washed with watertwice and washed with brine and dried over Na₂SO₄. Then concentrated togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 32monomer (10.5 g, 14.5 mmol, 75.9%) as a white solid. ESI-LCMS: m/z 776.3[M+H], ¹H-NMR (400 MHz, DMSO-d6): δ 11.40 (d, J=12.2 Hz, 1H), 8.04-7.96(dd, 1H), 7.43-7.24 (m, 9H), 6.92-6.87 (m, 4H), 5.84 (m, 1H), 4.93 (m,1H), 4.13 (m, 1H), 3.91-3.39 (m, 17H), 2.82 (t, J=5.6 Hz, 1H), 2.68 (t,J=6.0 Hz, 1H), 1.22-0.97 (m, 12H); ³¹P-NMR (162 MHz, DMSO-d6): δ 165.06,157.59.

Example 33. Synthesis of 5′ End Cap Monomer

Preparation of (2): To a solution of 1(11.2 g, 24.7 mmol) in DCM (120mL), imidazole (4.2 g, 61.9 mmol) and TBSCl (5.6 g, 37.1 mmol) wereadded at r.t., mixture was stirred at r.t. for 15 hrs, LCMS showed 1 wasconsumed completely. Mixture was added water (500 mL) and extracted withDCM (50 mL*2). The organic phase was dried over Na₂SO₄ and concentratedto give 2 (16.0 g) as an oil for the next step.

Preparation of (3): To a solution of 2 (16.0 g, 28.4 mmol) was added 6%DCA in DCM (160 mL) and triethylsilane (40 mL) at r.t. The reactionmixture was stirred at r.t. for 2 hrs. TLC showed 2 was consumedcompletely. Water (300 mL) was added, mixture was extracted with DCM (50mL*4), organic phase was dried by Na₂SO₄, concentrated by reducepressure to give crude which was purified by column chromatography(SiO₂, PE/EA=10:1 to 1:1) to give 3 (4.9 g, 65.9% yield) as an oil.ESI-LCMS: m/z 263 [M+H]⁺;¹H-NMR (400 MHz, DMSO-d6) δ 4.84-4.50 (m, 1H),4.3-4.09 (m, 1H), 3.90-3.80 (m, 1H), 3.75-3.67 (m, 1H), 3.65-3.57 (m,2H), 3.50-3.44 (m, 1H), 3.37-3.28 (m, 4H), 0.95-0.78 (s, 9H), 0.13-0.03(s, 6H).

Preparation of (4): To a solution of 3 (3.3 g, 12.6 mmol) in DMSO (33mL) was added EDCI (7.2 g, 37.7 mmol).The mixture was added pyridine(1.1 g, 13.8 mmol) and TFA (788.6 mg, 6.9 mmol). The reaction mixturewas stirred at r.t. for 3 hrs. TLC (PE/EA=4:1) showed 3 was consumed.The mixture was diluted with EA and water was added. The product wasextracted with EA. The organic layer was washed with brine and driedover Na₂SO₄ and concentrated to give the crude. This resulted in to give4 (3.23 g) as an oil for the next step.

Preparation of (5): To a solution of 4 (3.3 g, 12.6 mmol) in toluene (30mL) was added POM ester 4a (reference for 4a Journal of MedicinalChemistry, 2018, 61 (3), 734-744) (7.9 g, 12.6 mmol) and KOH (1.3 g,22.6 mmol) at r.t. The reaction mixture was stirred at 40° C. for 8 hrs.LCMS showed 4 was consumed. The mixture was diluted with water and EAwas added. The product was extracted with EA. The organic layer waswashed with brine and dried over Na₂SO₄ and concentrated to give thecrude. The crude was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=91/9 Detector, UV 254 nm. This resulted in to give 5 (5.4 g,9.5 mmol, 75.9% yield) as an oil. ESI-LCMS: m/z 567.2 [M+H]⁺; ¹H-NMR(400 MHz, CDCl₃) δ 6.89-6.77 (m, 1H), 6.07-5.96 (m, 1H), 5.86-5.55 (m,4H), 4.85-4.73 (m, 1H), 4.36-4.27 (m, 1H), 4.05-3.96 (m, 1H), 3.95-3.85(m, 1H), 3.73-3.65 (m, 1H), 3.44-3.35 (m, 3H), 1.30-1.25 (s, 18H),0.94-0.84 (s, 9H), 0.14-0.05 (s, 6H). ³¹P-NMR (162 MHz, CDCl₃) δ 18.30,15.11.

Preparation of (6): To a solution of 5 (5.4 g, 9.5 mmol) in HCOOH (30mL)/H₂O (30 mL)=1:1 at r.t. The reaction mixture was stirred at r.t. for15 hrs. LCMS showed the reaction was consumed. The mixture was dilutedwith con. NH₄OH till pH=7.5. The product was extracted with EA. Theorganic layer was washed with brine and dried over Na₂SO₄ andconcentrated to give the crude. The crude was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% HCOOH)=30/70 increasing toCH₃CN/H₂O (0.5% HCOOH)=70/30 within 45 min, the eluted product wascollected at CH₃CN/H₂O (0.5% HCOOH)=59/41 Detector, UV 220 nm. Thisresulted in to give 6 (2.4 g, 5.7 mmol, 59.4% yield) as an oil.ESI-LCMS: m/z 453.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6) δ 6.84-6.68 (m,1H), 6.07-5.90 (m, 1H), 5.64-5.55 (m, 4H), 5.32-5.24 (m, 1H), 4.23-4.15(m, 1H), 4.00-3.90 (m, 1H), 3.89-3.80 (m, 1H), 3.78-3.69 (m, 2H),3.37-3.30 (s, 3H), 1.30-1.10 (s, 18H). ³¹P-NMR (162 MHz, DMSO-d6) δ18.14.

Preparation of Example 33 monomer: To a solution of 6 (2.1 g, 4.5 mmol)in DCM (21 mL) were added DCI (452.5 mg, 3.8 mmol) and CEP[N(iPr)₂]₂(1.8 g, 5.9 mmol) at r.t. The reaction mixture was stirred at r.t. for15 hrs under N₂ atmosphere. LCMS showed 6 was consumed. The mixture wasdiluted with water. The product was extracted with DCM (30 mL). Theorganic layer was washed with brine and dried over Na₂SO₄ andconcentrated to give the crude. The crude was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 28 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=80/20 Detector, UV 254 nm. Thisresulted in to give Example 33 monomer (2.8 g, 4.3 mmol, 95.2% yield) asan oil. ESI-LCMS: m/z 653.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6) δ6.89-6.77 (m, 1H), 6.11-5.96 (m, 1H), 5.65-5.50 (m, 4H), 4.39-4.34 (d,J=20 Hz, 1H), 4.18-3.95 (m, 2H), 3.94-3.48 (s, 6H), 3.40-3.28 (m, 4H),2.84-2.75 (m, 2H), 1.26-1.98 (s, 30H). ³¹P-NMR (162 MHz, DMSO-d6) δ149.018, 148.736, 17.775, 17.508.

Example 34. Synthesis of 5′ End Cap Monomer

Preparation of (2): To a solution of 1 (ref for 1 Tetrahedron, 2013, 69,600-606) (10.60 g, 47.32 mmol) in DMF (106 mL), imidazole (11.26 g,165.59 mmol) and TBSCI (19.88 g, 132.53 mmol) were added. The mixturewas stirred at r.t. for 3.5 hrs, LCMS showed 1 was consumed completely.Water was added and extracted with EA, dried over by Na₂SO₄. Thefiltrate was evaporated under reduced pressure to give 2 (20.80 g, 45.94mmol, 97.19% yield) for the next step.

Preparation of (3): To a solution of 2 (20.80 g, 45.94 mmol) in THF (248mL), was added TFA (124 mL) and H₂O (124 mL) at 0° C., reaction mixturewas stirred for 30 min. LCMS showed 2 was consumed completely. Then wasextracted with EA, washed with sat. NaCl (aq.), dried over by Na₂SO₄.The filtrate was evaporated under reduced pressure to give the crudeproduct which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give 3 (10.00 g,29.59 mmol, 64.31% yield). ¹H-NMR (400 MHz, DMSO-d6): δ 7.33-7.18 (m,5H), 4.83-4.80 (m, 1H), 4.61-4.59 (m, 1H), 4.21-4.19 (m, 1H), 3.75-3.74(m, 1H), 3.23 (m, 3H), 3.13 (m, 3H), 2.41-2.40 (m, 1H), 0.81 (m, 9H),0.00 (m, 6H).

Preparation of (4): To a solution of 3(3.70 g, 10.95 mmol) in DMSO (37mL) was added EDCI (6.30 g, 32.84 mmol). Then pyridine (0.95 g, 12.05mmol) and TFA (0.69 g, 6.02 mmol) was added in N₂ atmosphere. Themixture was stirred for 3 hrs at r.t. LCMS showed 3 was consumedcompletely. Water was poured into and extracted with EA, washed withsat. NaCl (aq.), dried over by Na₂SO₄. The filtrate was evaporated underreduced pressure to give the crude product which was directly used fornext step.

Preparation of (5): To a solution of 4 in toluene (100.00 mL), was added4a (6.93 g, 10.97 mmol) and KOH (1.11 g, 19.78 mmol). It was stirred for3.5 hrs at 40° C. in N₂ atmosphere. TLC and LCMS showed 4 was consumedcompletely. Then was extracted with EA, washed with water and sat. NaCl(aq.), dried over by Na₂SO₄. The filtrate was evaporated under reducedpressure to give the crude product which was purified by Flash-Prep-HPLCwith the following conditions (IntelFlash-1): Column, C18 silica gel;mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 20 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in togive 5 (4.30 g, 6.70 mmol, 61.17% yield). ¹H-NMR (400 MHz, CDCl₃): δ7.27-7.26 (m, 4H), 7.17 (m, 1H), 6.94-6.82 (m, 1H), 6.13-6.02 (m, 1H),5.63-5.56 (m, 4H), 4.90-4.89 (m, 1H), 4.45-4.41 (m, 1H), 3.98-3.95 (m,1H), 3.39-3.29 (m, 4H), 1.90 (m, 1H), 1.12-0.83 (m, 29H), 0.00 (m, 7H);³¹P-NMR (162 MHz, CDCl₃): δ 18.021, 14.472.

Preparation of (6): To a solution of 5 (4.30 g, 6.70 mmol) in THF (43.00mL) was added HCOOH (100 mL) and H₂O (100 mL). It was stirred overnightat r.t. LCMS showed 5 was consumed completely. NH₄OH was poured into itand was extracted with EA, washed with sat. NaCl (aq.), dried over byNa₂SO₄. The filtrate was evaporated under reduced pressure to give thecrude product which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give 6 (2.10 g,3.98 mmol, 59.32% yield). ¹H-NMR (400 MHz, CDCl₃): δ 7.40-7.28 (m, 5H),7.11-7.00 (m, 1H), 6.19-6.14 (m, 1H), 5.71-5.68 (m, 4H), 4.95-4.94 (m,1H), 4.48-4.47 (m, 1H), 4.05-4.03 (m, 1H), 3.62-3.61 (m, 1H), 3.46 (m,3H), 3.00-2.99 (m, 1H), 1.22 (m, 18H); ³¹P-NMR (162 MHz, CDCl₃): δ18.134.

Preparation of Example 34 monomer: To a solution of 6 (2.10 g, 3.98mmol) in DCM (21 mL) was added DCI (410 mg, 3.47 mmol). CEP (1.40 g,4.65 mmol) was added in a N₂ atmosphere. LCMS showed 6 was consumedcompletely. DCM and H₂O was poured, the organic phase was washed withwater and sat. NaCl (aq.), dried over by Na₂SO₄. The filtrate wasevaporated under reduced pressure at 40° C. to give the crude productwhich was purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give Example 34 monomer (2.10g, 2.88 mmol). ¹H-NMR (400 MHz, DMSO-d6): δ 7.39-7.32 (m, 6H), 6.21-6.11(m, 1H), 5.64-5.61 (m, 4H), 4.91-4.85 (m, 1H), 4.59 (m, 1H), 4.28-4.25(m, 1H), 3.84-3.60 (m, 5H), 3.36-3.36 (m, 2H), 2.83-2.79 (m, 2H),1.18-1.14 (m, 29H); ³¹P-NMR (162 MHz, DMSO-d6): δ 149.588, 148.920,17.355, 17.010.

Example 35. Synthesis of 5′ End Cap Monomer

Preparation of (2): To a solution of 1 (5.90 g, 21.50 mmol) in DMF(60.00 mL), imidazole (4.39 g, 64.51 mmol) and TBSCl (7.63 g, 49.56mmol) were added. The mixture was stirred at r.t. for 3.5 hrs, LCMSshowed 1 was consumed completely. Water was added and extracted with EA,dried over by Na₂SO₄. The filtrate was evaporated under reduced pressureto give 2 (11.00 g, 21.91 mmol, 98.19% yield) for the next step.ESI-LCMS: m/z 225.1 [M+H]⁺.

Preparation of (3): To a solution of 2 (11.00 g, 21.91 mmol) in THF(55.00 mL) was added TFA (110.00 mL) and H₂O (55.00 mL) at 0° C.,reaction mixture was stirred for 30 min. LCMS showed 2 was consumedcompletely. Then was extracted with EA, washed with sat. NaCl (aq.),dried over by Na₂SO₄. The filtrate was evaporated under reduced pressureto give the crude product which was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 20 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in togive 3 (6.20 g, 16.32 mmol, 72.94% yield). ESI-LCMS: m/z 411.2 [M+H]⁺.

Preparation of (4): To a solution of 3 (3.50 g, 9.02 mmol) in DMSO(35.00 mL) was added EDCI (5.19 g, 27.06 mmol). Then pyridine (0.78 g,9.92 mmol) and TFA (0.57 g, 4.96 mmol) was added in N₂ atmosphere. Themixture was stirred for 3 h at r.t. Water was poured into it and wasextracted with EA, washed with sat. NaCl (aq.), dried over by Na₂SO₄.The filtrate was evaporated under reduced pressure to give the crudeproduct which was directly used for next step. ESI-LCMS: m/z 406.2[M+H]⁺.

Preparation of (5): To a solution of 4 in toluene (100.00 mL) was added4a (5.73 g, 9.07 mmol) and KOH (916.3 g, 16.33 mmol). It was stirred for3.5 h at 40° C. in N₂ atmosphere. Then was extracted with EA, washedwith water and sat. NaCl (aq.), dried over by Na₂SO₄. The filtrate wasevaporated under reduced pressure to give the crude product which waspurified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give 5 (5.02 g, 7.25 mmol,80.44% yield). ESI-LCMS: m/z 693.2 [M+H]⁺; ³¹P-NMR (162 MHz, DMSO-d6): δ17.811

Preparation of (6): To a solution of 5 (4.59 g, 6.63 mmol) in THF (46.00mL) was added HCOOH (92.00 mL) and H₂O (92.00 mL). It was stirredovernight at r.t. NH₄OH was poured into it and extracted with EA, washedwith sat. NaCl (aq.), dried over by Na₂SO₄. The filtrate was evaporatedunder reduced pressure to give the crude product which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give 6 (2.52 g, 4.36 mmol, 65.80% yield).

Preparation of Example 35 monomer: To a solution of 6 (2.00 g, 3.46mmol) in DCM (21.00 mL) was added DCI (370.00 mg, 3.11 mmol) and CEP(1.12 g, 4.15 mmol) was added in N₂ atmosphere. DCM and H₂O was poured,the organic phase was washed with water and sat. NaCl (aq.), dried overby Na₂SO₄. The filtrate was evaporated under reduced pressure at 38° C.to give the crude product which was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 20 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in togive Example 35 monomer (2.10 g, 2.70 mmol, 78.07% yield). ¹H-NMR (400MHz, DMSO-d6): δ 7.39-7.32 (m, 6H), 6.21-6.11 (m, 1H), 5.64-5.61 (m,4H), 4.91-4.85 (m, 1H), 4.59 (m, 1H), 4.28-4.25 (m, 1H), 3.84-3.60 (m,5H), 3.36-3.36 (m, 2H), 2.83-2.79 (m, 2H), 1.18-1.14 (m, 29H). ³¹P-NMR(162 MHz, DMSO-d6): δ 149.588, 148.920, 17.355, 17.010.

Example 36. Synthesis of Monomer

Preparation of(2): To a solution of 1(35.0 g, 53.2 mmol) in DMF (350 mL)was added imidazole (9.0 g, 133.0 mmol) then added TBSCl (12.0 g, 79.8mmol) at 0° C. The mixture was stirred at r.t. for 14 hrs. TLC showed 1was consumed completely. Water was added to the reaction. The productwas extracted with EA, The organic layer was washed with NaHCO₃ andbrine. Then the solution was concentrated under reduced pressure thecrude 2 (41.6 g) as a white solid which was used directly for next step.ESI-LCMS: m/z 772 [M+H]⁺.

Preparation of (3): To a solution of 2(41.0 g, 53.1 mmol) in 3% DCA(53.1 mmol, 350 mL) and Et₃SiH (53.1 mmol, 100 mL) at 0° C. The mixturewas stirred at 0° C. for 0.5 h. TLC showed 2 was consumed completely.NaHCO₃ was added to the reaction. The product was extracted with EA, Theorganic layer was washed with NaHCO₃ and brine. Then the solution wasconcentrated under reduced pressure. The residue silica gel columnchromatography (eluent, DCM/MeOH=100:1-20:1). This resulted in to give 3(20.0 g, 41.7 mmol, 78.6% over two step) as a white solid. ESI-LCMS: m/z470 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 12.12 (s, 1H), 11.67 (s, 1H),8.28 (s, 1H), 6.12-6.07 (dd, J=15 Hz, 1H), 5.75 (d, J=5 Hz, 1H),5.48-5.24 (m, 2H), 4.55-4.49 (m, 1H), 3.97 (s, 1H), 3.75-3.55 (m, 2H),2.79-2.76 (m, 1H), 1.12 (d, J=6 Hz, 6H), 0.88 (s, 9H), 0.11 (d, J=6 Hz,6H).

Preparation of (4): To the solution of 3 (20 g, 42.6 mmol) in dry DCM(100 mL) and DMF (60 mL) was added PDC (20. g, 85.1 mmol), tert-butylalcohol (63.1 g, 851.8 mmol) and Ac₂O (43.4 g, 425.9 mmol) at r.t. underN₂ atmosphere. And the reaction mixture was stirred at r.t. for 2 h. Thesolvent was removed to give a residue which was purified by silica gelcolumn chromatography (eluent, PE: EA=4:1-2:1) to give a residue whichwas purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give 4 (16.0 g, 29.0 mmol,68.2% yield) as a white solid. ESI-LCMS: m/z 540 [M+H]⁺; H-NMR (400 MHz,DMSO-d6): δ 12.12 (s, 1H), 11.69 (s, 1H), 8.28 (s, 1H), 6.21-6.17 (dd,J=15 Hz, 1H), 5.63-5.55 (m, 1H), 4.75-4.72 (m, 1H), 4.41 (d, J=5 Hz,1H), 2.79-2.76 (m, 1H), 1.46 (s, 9H), 1.13-1.11 (m, 6H), 0.90 (s, 9H),0.14 (d, J=2 Hz, 6H).

Preparation of (5): To the solution of 4 (16.0 g, 29.6 mmol) in dryTHF/MeOD/D20=10/2/1 (195 mL) was added NaBD₄ (3.4 g, 88.9 mmol) at r.t.and the reaction mixture was stirred at 50° C. for 2 h. After completionof reaction, adjusted pH value to 7 with CH₃COOD, after addition ofwater, the resulting mixture was extracted with EA (300 mL). Thecombined organic layer was washed with water and brine, dried overNa₂SO₄, Then the solution was concentrated under reduced pressure thecrude 5 (11.8 g) as a white solid which was used directly for next step.ESI-LCMS: m/z 402 [M+H]⁺.

Preparation of (6): To a solution of 5 (5.0 g, 12.4 mmol) in pyridine(50 mL) was added iBuCl (2.6 g, 24.9 mmol) at 0° C. under N₂ atmosphere.The mixture was stirred at r.t. for 14 h. TLC showed 5 was consumedcompletely. Then the solution diluted with EA. The organic layer waswashed with NaHCO₃ and brine. Then the solution was concentrated underreduced pressure to give the crude. To a solution of the crude inpyridine (50 mL) was added 2N NaOH (MeOH/H₂O=4:1, 15 mL) at 0° C. Themixture was stirred at 0° C. for 10 min. Then the solution diluted withEA. The organic layer was washed with NH₄Cl and brine. Then the solutionwas concentrated under reduced pressure the residue was purified byFlash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=4/1 within 25 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=3/2; Detector, UV 254 nm. Thisresulted in to give 6 (6 g, 10.86 mmol, 87.17% yield) as a white solid.ESI-LCMS: m/z 472.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 12.12 (s, 1H),11.67 (s, 1H), 8.28 (s, 1H), 6.12-6.07 (dd, J=15 Hz, 1H), 5.48-5.24 (m,2H), 5.22 (s, 1H), 4.55-4.49 (m, 1H), 3.97 (d, J=5 Hz, 1H), 2.79-2.76(m, 1H), 1.12 (d, J=6 Hz, 6H), 0.88 (s, 9H), 0.11 (d, J=6 Hz, 6H).

Preparation of (7): To a solution of 6 (3.8 g, 8.1 mmol) in pyridine (40mL) was added DMTrCl (4.1 g, 12.1 mmol) at 20° C. The mixture wasstirred at 20° C. for 1 h. TLC showed 7 was consumed completely. Waterwas added to the reaction. The product was extracted with EA, Theorganic layer was washed with NaHCO₃ and brine. Then the solution wasconcentrated under reduced pressure to give the crude product of 7 (6 g,7.6 mmol, 94.3% yield) as a yellow solid. ESI-LCMS: m/z 775 [M+H]⁺.

Preparation of (8): To a solution of 7 (6.0 g, 7.75 mmol) in THF (60 mL)was added TBAF (2.4 g, 9.3 mmol). The mixture was stirred at r.t. for 1h. TLC showed 7 was consumed completely. Water was added to thereaction. The product was extracted with EA, The organic layer waswashed with NaHCO₃ and brine. Then the solution was concentrated underreduced pressure, the residue was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 25 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=4/1; Detector, UV 254 nm. This resulted in togive 8 (4.0 g, 5.9 mmol, 76.6% yield) as a white solid. ESI-LCMS: m/z660 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 12.12 (s, 1H), 11.67 (s, 1H),8.12 (s, 1H), 7.34-7.17 (m, 9H), 6.83-6.78 (m, 4H), 6.23-6.18 (m, 1H),5.66 (d, J=7 Hz, 1H), 5.48-5.35 (m, 1H), 4.65-4.54 (m, 1H), 3.72 (d, J=2Hz, 6H), 2.79-2.73 (m, 1H), 1.19-1.06 (m, 6H).

Preparation of Example 36 monomer: To a solution of 9 (4.0 g, 6.1 mmol)in DCM (40 mL) was added DCI (608 mg, 5.1 mmol) and CEP (2.2 g, 7.3mmol) under N₂ pro. The mixture was stirred at 20° C. for 0.5 h. TLCshowed 9 was consumed completely. The product was extracted with DCM,The organic layer was washed with H₂O and brine. Then the solution wasconcentrated under reduced pressure and the residue was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give Example 36 monomer (5.1 g, 5.81 mmol, 95.8% yield)as a white solid. ESI-LCMS: m/z 860 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ12.12 (s, 1H), 11.67 (s, 1H), 8.12 (s, 1H), 7.34-7.17 (m, 9H), 6.83-6.78(m, 4H), 6.23-6.18 (m, 1H), 5.67-5.54 (m, 1H), 4.70-4.67 (m, 1H),4.23-4.20 (m, 1H), 3.72 (m, 6H), 3.60-3.48 (m, 3H), 2.79-2.58 (m, 3H),1.13-0.94 (m, 18H); ³¹P-NMR (162 MHz, DMSO-d6): δ 150.31, 150.26,140.62, 149.57.

Example 37: Synthesis of Monomer

Preparation of (2): To a solution of 1(35 g, 130.2 mmol) in DMF (350 mL)was added imidazole (26.5 g, 390.0 mmol) then added TBSCl (48.7 g, 325.8mmol) at 0° C. The mixture was stirred at r.t. for 14 h. TLC showed 1was consumed completely. Water was added to the reaction. The productwas extracted with EA, The organic layer was washed with NaHCO₃ andbrine. Then the solution was concentrated under reduced pressure thecrude 2 (64.6 g) as a white solid which was used directly for next step.ESI-LCMS: m/z 498 [M+H]⁺.

Preparation of (3): To a solution of 2 (64.6 g, 130.2 mmol) in THF (300mL) and added TFA/H₂O (1:1, 300 mL) at 0° C. The mixture was stirred at0° C. for 2 h. TLC showed 2 was consumed completely. NaHCO₃ was added tothe reaction. The product was extracted with EA, The organic layer waswashed with NaHCO₃ and brine. Then the solution was concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (eluent, DCM: MEOH=100:1-20:1). This resulted in to give3 (31.3 g, 81.7 mmol, 62.6% over two step) as a white solid. ESI-LCMS:m/z 384 [M+H]⁺.

Preparation of (4): To a solution of 3 (31.3 g, 81.7 mmol) in ACN/H₂O(1:1, 350 mL) was added DAIB (78.0 g, 244.0 mmol) and Tempo (3.8 g, 24.4mmol). The mixture was stirred at 40° C. for 2 h. TLC showed 3 wasconsumed completely. Then filtered to give 4 (22.5 g, 55.5 mmol, 70.9%)as a white solid. ESI-LCMS: m/z 398 [M+H]⁺.

Preparation of (5): To a solution of 4 (22.5 g, 55.5 mmol) in MeOH (225mL) held at −15° C. with an ice/MeOH bath was added SOCl₂ (7.6 mL, 94.5mmol), dropwise at such a rate that the reaction temp did not exceed 7°C. After the addition was complete, cooling was removed, the reactionwas allowed to stir at room temp. The mixture was stirred at r.t. for 14h. TLC showed 4 was consumed completely. Then the solution wasconcentrated under reduced pressure to get crude 5 (23.0 g) as a whitesolid which was used directly for next step. ESI-LCMS: m/z 298 [M+H]⁺.

Preparation of (6): To a solution of 5 (23 g, 55.5 mmol) in DMF (220 mL)was added imidazole (11.6 g, 165.0 mmol) then added TBSCI (12.3 g, 82.3mmol) at 0° C. The mixture was stirred at 20° C. for 14 h. TLC showed 1was consumed completely. Water was added to the reaction. The productwas extracted with EA, The organic layer was washed with NaHCO₃ andbrine. Then the solution was concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (eluent, DCM:MEOH=100:1-20:1). This resulted in to give 6 (21.3 g, 51.1 mmol, 90%over two step) as a white solid. ESI-LCMS: m/z 412 [M+H]⁺.

Preparation of (7): To the solution of 6 (21.0 g, 51.0 mmol) in dryTHF/MeOD/D20=10/2/1 (260.5 mL) was added NaBD₄ (6.4 g, 153.1 mmol) atr.t. and the reaction mixture was stirred at 50° C. for 2 h. Aftercompletion of reaction, the resulting mixture was added CH₃COOD to pH=7,after addition of water, the resulting mixture was extracted with EA(300 mL). The combined organic layer was washed with water and brine,dried over Na₂SO₄. Then the solution was concentrated under reducedpressure and the residue was used for next step without furtherpurification. ESI-LCMS: m/z 386 [M+H]⁺.

Preparation of (8): To a stirred solution of 7 (14.0 g, 35 mmol) inpyridine (50 mL) were added BzCl (17.2 g, 122.5 mmol) at 0° C. under N₂atmosphere. The mixture was stirred at r.t. for 14 h. TLC showed 7 wasconsumed completely. Then the solution diluted with EA. The organiclayer was washed with NaHCO₃ and brine. Then the solution wasconcentrated under reduced pressure and the residue was used for nextstep without further purification. To a solution of the crude inpyridine (300 mL) then added 2M NaOH (MeOH: H₂O=4:1, 60 mL) at 0° C. Themixture was stirred at 0° C. for 10 min. Then the solution diluted withEA. The organic layer was washed with NH₄Cl and brine. Then the solutionwas concentrated under reduced pressure and the residue was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=4/1 within 25 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=3/2; Detector, UV 254 nm. Thisresulted in to give 8 (14 g, 28.02 mmol, 69.21% yield) as a white solid.ESI-LCMS: m/z 490 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.24 (s, 1H),8.76 (s, 1H), 8.71 (m, 1H), 8.04 (d, J=7 Hz, 2H), 7.66-7.10 (m, 5H),6.40-6.35 (dd, 1H), 5.71-5.56 (m, 1H), 5.16 (s, 1H), 4.79-4.72 (m, 1H),4.01 (m, 1H), 0.91 (s, 9H), 0.14 (m, 6H).

Preparation of (9): To a solution of 8 (5.1 g, 10.4 mmol) in pyridine(50 mL) was added DMTrC1 (5.3 g, 15.6 mmol). The mixture was stirred atr.t. for 1 h. TLC showed 8 was consumed completely. Water was added tothe reaction. The product was extracted with EA, The organic layer waswashed with NaHCO₃ and brine. Then the solution was concentrated underreduced pressure and the residue was used for next step without furtherpurification. ESI-LCMS: m/z 792 [M+H]⁺.

Preparation of (10): To a solution of 9 (7.9 g, 10.0 mmol) in THF (80mL) was added 1M TBAF in THF (12 mL). The mixture was stirred at r.t.for 1 h. TLC showed 9 was consumed completely. Water was added to thereaction. The product was extracted with EA, The organic layer waswashed with NaHCO₃ and brine. Then the solution was concentrated underreduced pressure the residue was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 25 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=4/1; Detector, UV 254 nm. This resulted in togive 10 as a white solid. ESI-LCMS: m/z 678 [M+H]; ¹H-NMR (400 MHz,DMSO-d6): δ 11.25 (s, 1H), 8.74 (s, 1H), 8.62 (s, 1H), 8.04 (d, J=7 Hz,2H), 7.66-7.53 (m, 3H), 7.33-7.15 (m, 9H), 6.82-6.78 (m, 4H), 6.43 (d,J=20 Hz, 1H), 5.76-5.60 (m, 1H), 4.88-4.80 (m, 1H), 4.13 (d, J=8 Hz,1H), 3.71 (m, 6H).

Preparation of Example 37 monomer: To a solution of 10 (6.2 g, 9.1 mmol)in DCM (60 mL) was added DCI (1.1 g, 9.4 mmol) and CEP (3.3 g, 10.9mmol) under N₂ pro. The mixture was stirred at 20° C. for 0.5 h. TLCshowed 10 was consumed completely. The product was extracted with DCM,The organic layer was washed with H₂O and brine. Then the solution wasconcentrated under reduced pressure and the residue was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give Example 37 monomer (7.5 g, 8.3 mmol, 90.7%) as awhite solid. ESI-LCMS: m/z 878 [M+H]+; ¹H-NMR (400 MHz, DMSO-d6): δ11.25 (s, 1H), 8.68-8.65 (dd, 2H), 8.04 (m, 2H), 7.66-7.53 (m, 3H),7.33-7.15 (m, 9H), 6.82-6.78 (m, 4H), 6.53-6.43 (m, 1H), 5.96-5.81 (m,1H), 5.36-5.15 (m, 1H), 4.21 (m, 1H), 3.86-3.52 (m, 10H), 2.79-2.61 (m,2H), 1.21-0.99 (m, 12H); ³¹P-NMR (162 MHz, DMSO-d6): δ 149.60, 149.56,149.48.

Example 38. Synthesis of End Cap Monomer

Preparation of (2): To a solution of 1(20.0 g, 71.2 mmol) in drypyridine (200.0 mL) was added TBSCl (26.8 g, 177.9 mmol) and imidazole(15.6 g, 227.8 mmol). The mixture was stirred at r.t. for 15 h. TLCshowed 1 was consumed completely. The reaction mixture was concentratedto give residue. The residue was quenched with DCM (300.0 mL). The DCMlayer was washed with H₂O (100.0 mL*2) and brine. The DCM layerconcentrated to give crude 2 (45.8 g) as a yellow oil. The crude used tonext step directly. ESI-LCMS m/z 510.5 [M+H]⁺.

Preparation of (3): To a mixture solution of 2 (45.8 g) in THF (300.0mL) was added mixture of H₂O (100.0 mL) and TFA (100.0 mL) at 0° C. over30 min. Then the reaction mixture was stirred at 0° C. for 4 h. TLCshowed the 2 was consumed completely. The reaction mixture pH wasadjusted to 7-8 with NH₃.H₂O (100 mL). Then the mixture was extractedwith EA (500.0 mL*2). The combined EA layer was washed with brine andconcentrated to give crude which was purified by c.c. (PE:EA=5:1-1:0) togive compound 3 (21.0 g, 53.2 mmol, 74.7% yield over 2 steps) as a whitesolid. ESI-LCMS m/z 396.2 [M+H]⁺.

Preparation of (4): To a solution of 3 (21.0 g, 53.2 mmol) in ACN (100.0mL) and water (100.0 mL) were added (diacetoxyiodo)benzene (51.0 g,159.5 mmol) and TEMPO (2.5 g, 15.9 mmol), The reaction mixture wasstirred at 40° C. for 1 h. TLC showed the 3 was consumed completely. Thereaction mixture was cooled down to r.t. and filtered, the filtrate wasconcentrated to give crude which was purified by crystallization (ACN)to give 4 (14.5 g, 35.4 mmol, 66.2% yield). ESI-LCMS m/z 410.1[M+H]⁺.

Preparation of (5): To a solution of 4 (14.5 g, 35.4 mmol) in toluene(90.0 mL) and MeOH (60.0 mL) was added trimethylsilyldiazomethane (62.5mL, 2.0 M, 141.8 mmol) at 0° C., then stirred at r.t. for 2 h. TLCshowed the 4 was consumed completely. The solvent was removed underreduce pressure, the residue was purified by crystallization (ACN) togive 5 (10.0 g, 23.6 mmol, 66.6% yield). ESI-LCMS m/z 424.2 [M+H]⁺

Preparation of (6): To the solution of 5 (10.0 g, 23.6 mmol) in dryTHF/MeOD/D20=10/2/1 (100.0 mL) was added NaBD₄ (2.98 g, 70.9 mmol) threetimes during an hour at 40° C., the reaction mixture was stirred at r.t.for 2.0 h. The resulting mixture was added CH₃COOD change pH=7.5, afteraddition of water, the resulting mixture was extracted with EA (50.0mL*3). The combined organic layer was washed with water and brine, driedover Na₂SO₄, concentrated to give a residue which was purified by c.c.(PE/EA=1:1-1:0). This resulted in to give 6 (6.1 g, 15.4 mmol, 65.3%yield) as a white solid. ESI-LCMS m/z 398.1 [M+H]⁺; ¹H-NMR (400 MHz,DMSO-d6) δ 8.28 (s, 1H), 8.02 (s, 1H), 7.23 (s, 2H), 5.86 (d, J=6.4 Hz,1H), 5.26 (s, 1H), 4.42-4.41 (m, 1H), 4.35-4.32 (m, 1H), 3.82 (d, J=2.6Hz, 1H), 3.14 (s, 3H), 0.78 (s, 9H), 0.00 (d, J=0.9 Hz, 6H).

Preparation of (7): To a solution of 6 (6.1 g, 15.4 mmol) in pyridine(60.0 mL) was added the benzoyl chloride (6.5 g, 46.2 mmol) drop wise at5° C. The reaction mixture was stirred at r.t. for 2 h. TLC showed the 6was consumed completely. The reaction mixture was cooled down to 10° C.and quenched with H₂O (20.0 mL), extracted with EA (200.0 mL*2),combined the EA layer. The organic phase was washed with brine and driedover Na₂SO₄, concentrated to give the crude (12.0 g) which was dissolvedin pyridine (60.0 mL), cooled to 0° C., 20.0 mL NaOH (2 M in methanol:H₂O=4: 1) was added and stirred for 10 min. The reaction was quenched bysaturated solution of ammonium chloride, the aqueous layer was extractedwith EA (200.0 mL*2), combined the EA layer, washed with brine and driedover Na₂SO₄, concentrated. The residue was purified by c.c. (PE/EA=10:1˜1:1) to give 7 (7.0 g, 13.9 mmol, 90.2% yield). ESI-LCMS m/z 502.2[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H, exchanged with D20)8.77 (s, 2H), 8.04-8.06 (m, 2H), 7.64-7.66 (m, 2H), 7.54-7.58 (m, 2H),6.14-6.16 (d, J=5.9 Hz, 1H), 5.20-5.23 (m, 1H), 4.58-4.60 (m, 1H),4.52-4.55 (m, 1H), 3.99-4.01 (m, 1H), 3.34 (s, 4H), 0.93 (s, 9H),0.14-0.15 (d, J=1.44 Hz, 6H).

Preparation of (8): To a stirred solution of 7 (5.5 g, 10.9 mmol) inDMSO (55.0 mL) was added EDCI (6.3 g, 32.9 mmol), pyridine (0.9 g, 10.9mmol) and TFA(0.6 g,5.5 mmol), the reaction mixture was stirred at r.t.for 15 h. The reaction was quenched with water and extracted with EA(100.0 mL). The organic phase was washed by brine, dried over Na₂SO₄,The organic phase was evaporated to dryness under reduced pressure togive a residue 8 (4.8 g) which was used directly to next step. ESI-LCMS:m/z 517.1 [M+H₂O]⁺.

Preparation of (9b): A solution of 9a (35.0 g, 150.8 mmol) and NaI (90.5g, 603.4 mmol) in dry ACN (180.0 mL) was added chloromethyl pivalate(113.6 g, 754.3 mmol) at r.t., the reaction was stirred at 80° C. for 4h. The reaction was cooled to r.t. and quenched by water, then themixture was extracted with EA (500.0 mL *3), combined the organic layerwas washed with saturated solution of ammonium chloride, followed bywith brine and dried over Na₂SO₄. Then the organic layer wasconcentrated to give a residue which was purified by c.c., this resultedin to give 9b (38.0 g, 60.1 mmol, 39.8% yield) as a white solid.ESI-LCMS m/z 655.2 [M+Na]⁺; ¹H-NMR (400 MHz, CDCl₃): S 5.74-5.67 (m,8H), 2.67 (t, J=21.6 Hz, 2H), 1.23 (s, 36H).

Preparation of (9): 3.8 g 10% Pd/C was washed with dry THF (30.0 mL)three times. Then transferred into a round-bottom flask charged with 9b(38.0 g, 60.1 mmol) and solvent (dry THF:D₂O=5:1, 400.0 mL), the mixturewas stirred at 80° C. under 1 L H₂ balloon for 15 h. The reaction wascooled to r.t. and extracted with EA (500.0 mL *3), combined the organiclayer was washed with brine and dried over Na₂SO₄. The residue 9 (3.0 g,3.7 mmol, 38.8% yield) as a white solid was used directly to next stepwithout further purification. ESI-LCMS m/z 657.2 [M+Na]⁺; ¹H-NMR (400MHz, CDCl₃): δ 5.74-5.67 (m, 8H), 1.23 (s, 36H).

Preparation of (10): A solution of 8 (4.8 g, 9.6 mmol), 9 (7.3 g, 11.5mmol) and K₂CO₃(4.0 g, 38.8 mmol) in dry THF (60.0 mL) and D20 (20.0 mL)was stirred at r.t. 18 h. LC-MS showed 8 was consumed completely. Theproduct was extracted with EA (300.0 mL) and the organic layer waswashed with brine and dried over Na₂SO₄. Then the organic layer wasconcentrated to give a residue which was purified by c.c.(PE/EA=5:1-1:1) and MPLC. This resulted in to give 10 (3.0 g, 3.7 mmol,38.8% yield) as a white solid. ESI-LCMS m/z 806.4[M+H]⁺; ¹H-NMR (400MHz, DMSO-d6): δ 11.25 (s, 1H, exchanged with D20) 8.75 (s, 2H),8.07-8.05 (d, J=8.0 Hz, 2H), 7.67-7.54 (m, 3H), 6.05 (d, J=5.1 Hz, 1H),5.65-5.58 (m, 4H), 4.80-4.70 (m, 2H), 4.59-4.57 (m, 1H), 3.36 (s, 3H),1.11 (s, 9H), 1.10 (s, 9H), 0.94 (s, 9H), 0.17-0.16 (m, 6H); ³¹P NMR(162 MHz, DMSO-d6) δ 17.02.

Preparation of (11): To a round-bottom flask was added 10 (3.0 g, 3.7mmol) in a mixture of H₂O (30.0 mL), HCOOH (30.0 mL). The reactionmixture was stirred at 40° C. for 15 hrs. LC-MS showed the 10 wasconsumed completely. The reaction mixture was adjusted the pH=6-7 withcon. NH₃.H₂O (100.0 mL). Then the mixture was extracted with DCM (100.0mL*3). The combined DCM layer was dried over Na₂SO₄. Filtered andfiltrate was concentrated to give crude which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/2 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=3/2; Detector, UV 254 nm. To giveproduct 11 (1.8 g, 2.6 mmol, 70.3% yield). ESI-LCMS m/z=692.2[M+H]⁺;¹H-NMR (400 MHz, DMSO-d6): δ 11.11 (s, 1H, exchanged with D20) 8.71-8.75(d, J=14.4, 2H), 8.04-8.06 (m, 2H), 7.64-7.65 (m, 1H), 7.54-7.58 (m,2H), 6.20-6.22 (d, J=5.4, 2H), 5.74-5.75 (d, J=5.72, 2H), 5.56-5.64 (m,4H), 4.64-4.67 (m, 1H), 4.58-4.59 (m, 1H), 4.49-4.52 (m, 1H), 3.37 (s,3H), 1.09-1.10 (d, J=1.96, 18H); ³¹P NMR (162 MHz, DMSO-d6) δ 17.46.

Preparation of Example 38 monomer: To a solution of 11 (1.8 g, 2.6 mmol)in DCM (18.0 mL) was added the DCI (276.0 mg, 2.3 mmol), thenCEP[N(ipr)₂]₂ (939.5 mg, 3.1 mmol) was added. The mixture was stirred atr.t. for 1 h. TLC showed 11 consumed completely. The reaction mixturewas washed with H₂O (50.0 mL*2) and brine (50.0 mL*2), dried over Na₂SO₄and concentrated to give crude which was purified by Flash-Prep-HPLCwith the following conditions (IntelFlash-1): Column, C18 silica gel;mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 20 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=9/1; Detector, UV 254 nm. The product wasconcentrated to give Example 38 monomer (2.0 g, 2.2 mmol, 86.2% yield)as a white solid. ESI-LCMS m/z 892.3[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ11.27 (s, 1H, exchanged with D20) 8.72-8.75 (m, 2H), 8.04-8.06 (m, 2H),7.54-7.68 (m, 3H), 6.20-6.26 (m, 1H), 5.57-5.64 (m, 4H), 4.70-4.87 (m,3H), 3.66-3.88 (m, 4H), 3.37-3.41 (m, 3H), 2.82-2.86 (m, 2H), 1.20-1.21(m, 12H), 1.08-1.09 (m, 18H); ³¹P-NMR (162 MHz, DMSO-d6): δ 150.03,149.19, 17.05, 16.81.

Example 39. Synthesis of 5′ End Cap Monomer

Preparation of (6): To a stirred solution of 5 (8.08 g, 21.3 mmol,Scheme 3) in DMSO (80.0 mL) were added EDCI(12.2 g, 63.9 mmol),pyridine(1.7 g,21.3 mmol),TFA(1.2 g,10.6 mmol) at r.t. And the reactionmixture was stirred at r.t. for 1.5 h. The reaction was quenched withwater and extracted with EA (200.0 mL). The organic phase was washed bybrine, dried over Na₂SO₄, The organic phase was evaporated to drynessunder reduced pressure to give a residue 6 which was used directly tonext step. ESI-LCMS: m/z 372.3 [M+H]⁺.

Preparation of (8): To a solution of K₂CO₃ (5.5 g, 8.3 mmol) in dry THF(60.0 mL) and D20 (20.0 mL) was added a solution of 6 (8.0 g, 21.5 mmol)in dry THF(10.0 mL). The reaction mixture was stirred at r.t. overnight.LC-MS showed 6 was consumed completely. The product was extracted withEA (300.0 mL) and the organic layer was washed with brine and dried overNa₂SO₄. Then the organic layer was concentrated to give a residue whichwas purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1;Detector, UV 254 nm. This resulted in to give 8 (5.0 g, 7.3 mmol, 40.0%)as a white solid. ESI-LCMS: m/z 679.3 [M+H]⁺; ¹H-NMR (400 MHz,Chloroform-d): δ 9.91 (s, 1H), 7.29 (d, J=8.1 Hz, 1H), 5.82 (d, J=2.7Hz, 1H), 5.72 (d, J=8.1 Hz, 1H), 5.65-5.54 (m, 4H), 4.43 (dd, J=7.2, 3.2Hz, 1H), 3.92 (dd, J=7.2, 5.0 Hz, 1H), 3.65 (dd, J=5.1, 2.7 Hz, 1H),3.44 (s, 3H), 1.13 (s, 18H), 0.82 (s, 9H), 0.01 (d, J=4.8 Hz, 6H); ³¹PNMR (162 MHz, Chloroform-d): δ 16.40.

Preparation of (9): To a solution of HCOOH (50.0 mL) and H₂O (50.0 mL)was added 8 (5.0 g,7.3 mmol). The reaction mixture was stirred at 40° C.overnight. LC-MS showed 8 was consumed completely. A solution ofNaHCO₃(500.0 mL) was added. The product was extracted with EA (300.0 mL)and the organic layer was washed with brine and dried over Na₂SO₄. Thenthe organic layer was concentrated to give a residue which was purifiedby Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=3/2 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. Thisresulted in to give 9 (3.0 g, 5.4 mmol, 73.2%) as a white solid.ESI-LCMS: m/z 565.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.43 (s, 1H),7.64 (d, J=8.1 Hz, 1H), 5.83 (d, J=4.3 Hz, 1H), 5.69-5.56 (m, 5H), 5.54(d, J=6.7 Hz, 1H), 4.37 (dd, J=6.1, 2.9 Hz, 1H), 4.12 (q, J=6.1 Hz, 1H),3.96 (dd, J=5.4, 4.3 Hz, 1H), 3.39 (s, 3H), 1.16 (s, 18H); ³¹P NMR (162MHz, DMSO-d6): δ 17.16.

Preparation of Example 39 monomer: To a suspension of 9 (2.6 g, 4.6mmol) in DCM (40.0 mL) was added DCI (0.5 g, 5.6 mmol) and CEP[N(iPr)₂]₂(1.7 g, 5.6 mmol). The mixture was stirred at r.t. for 1.0 h. LC-MSshowed 9 was consumed completely. The solution was washed with watertwice and washed with brine and dried over Na₂SO₄. Then concentrated togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 39monomer (3.0 g, 3.9 mmol, 85.2%) as a white solid. . ESI-LCMS: m/z 765.3[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 11.44 (s, 1H), 7.71 (dd, J=8.1, 3.8Hz, 1H), 5.81 (dd, J=4.4, 2.5 Hz, 1H), 5.74-5.53 (m, 5H), 4.59-4.33 (m,2H), 4.20-4.14 (m, 1H), 3.88-3.53 (m, 4H), 3.39 (d, J=16.2 Hz, 3H), 2.80(td, J=5.9, 2.9 Hz,2 H), 1.16 (d, J=1.9 Hz, 30H); ³¹P-NMR (162 MHz,DMSO-d6): δ 147.68, 149.16, 16.84, 16.55.

Example 40. Synthesis of Monomer

Preparation of (2): To a solution of 1(26.7 g*2, 0.1 mol) in DMF (400mL) was added sodium hydride (4.8 g, 0.1 mol) for 30 min, then was addedCD3I (16 g, 0.1 mol) at 0° C. for 2.5 hr (ref. for selective2′-O-alkylation reaction conditions, J. Org. Chem. 1991, 56, 5846-5859).The mixture was stirring at r.t. for another 1 h. LCMS showed thereaction was consumed. The mixture was filtered and the clear solutionwas evaporated to dryness and was evaporated with CH₃₀H. The crude waspurified by silica gel column (SiO₂, DCM/MeOH=50:1-15:1). This resultedin to give the product 2 (35.5 g, 124.6 mmol, 62% yield) as a solid.ESI-LCMS: m/z 285 [M+H]⁺.

Preparation of (3): To a solution of 2 (35.5 g, 124.6 mmol) in pyridine(360 mL) was added imidazole (29.7 g, 436.1 mmol) and TBSCI (46.9 g,311.5 mmol). The mixture was stirred at r.t. over night. LCMS showed 2was consumed completely. The reaction was quenched with water (500 mL).The product was extracted into ethyl acetate (1 L). The organic layerwas washed with brine and dried over anhydrous Na₂SO₄. The crude waspurified by silica gel column (SiO₂, PE/EA=4:1-1:1). This resulted in togive the product 3 (20.3 g, 39.6 mmol, 31.8% yield) as a solid.ESI-LCMS: m/z 513 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 8.32 (m, 1H),8.13 (m, 1H), 7.31 (m, 2H), 6.02-6.01 (d, J=4.0 Hz, 1H), 4.60-4.58 (m,1H), 4.49-4.47 (m, 1H), 3.96-3.86 (m, 2H), 3.72-3.68 (m, 1H), 0.91-0.85(m, 18H), 0.13-0.01 (m, 12H).

Preparation of (4): To a solution of 3 (20.3 g, 39.6 mmol) in THF (80mL) was added TFA (20 mL) and water (20 mL) at 0° C. The reactionmixture was stirred at 0° C. for 5 h. LC-MS showed 3 was consumedcompletely. Con. NH₄OH was added to the mixture at 0° C. to quench thereaction until the pH=7.5. The product was extracted into ethyl acetate(200 mL). The organic layer was washed with brine and dried overanhydrous Na₂SO₄. The solution was then concentrated under reducedpressure and the residue was washed by PE/EA=5:1. This resulted in togive 4 (10.5 g, 26.4 mmol, 66.6% yield) as a white solid. ESI-LCMS: m/z399 [M+H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ 8.41 (m, 1H), 8.14 (m, 1H),7.37 (m, 2H), 5.99-5.97 (d, J=8.0 Hz, 1H), 5.43 (m, 1H), 4.54-4.44 (m,2H), 3.97-3.94 (m, 1H), 3.70-3.53 (m, 2H), 0.91 (m, 9H), 0.13-0.12 (m,6H).

Preparation of (5): To a solution of 4 (10.5 g, 26.4 mmol) inACN/H₂O=1:1 (100 mL) was added DAIB (25.4 g, 79.2 mmol) and TEMPO (1.7g, 7.9 mmol). The reaction mixture was stirred at 40° C. for 2 h. LCMSshowed 4 was consumed. The mixture was diluted with EA and water wasadded. The product was extracted with EA. The organic layer was washedwith brine and dried over anhydrous Na₂SO₄. The solution was thenconcentrated under reduced pressure and the residue was washed by ACN.This resulted in to give 5 (6.3 g, 15.3 mmol, 57.9% yield) as a whitesolid. ESI-LCMS: m/z 413 [M+H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ=8.48 (m,1H), 8.16 (m, 1H), 7.41 (m, 2H), 6.12-6.10 (d, J=8.0 Hz, 1H), 4.75-4.73(m, 1H), 4.42-4.36 (m, 2H), 3.17 (m, 6H), 2.07 (m, 2H), 0.93 (m, 9H),0.17-0.15 (m, 6H).

Preparation of (6): To a solution of 5 (6.3 g, 15.3 mmol) in toluene (36mL) and methanol (24 mL) was added (trimethylsilyl)diazomethane (7.0 g,61.2 mmol) till the yellow color not disappear at r.t. for 2 min. LCMSshowed the reaction was consumed. The solvent was removed to give thecured 6 (6.0 g) as a solid witch used for the next step. ESI-LCMS: m/z427 [M+H]+;¹H-NMR (400 MHz, DMSO-d6): δ 8.45 (m, 1H), 8.15 (m, 1H), 7.35(m, 2H), 6.12-6.10 (d, J=8.0 Hz, 1H), 4.83-4.81 (m, 1H), 4.50-4.46 (m,1H), 3.73 (m, 3H), 3.31 (m, 1H), 0.93 (m, 9H), 0.15-0.14 (m, 6H).

Preparation of (7): To the solution of 6 (6 g) in dryTHF/MeOD/D20=10/2/1 (78 mL) was added NaBD₄ (2.3 g, 54.8 mmol) at r.t.And the reaction mixture was stirred at r.t for 2.5 hr. After completionof reaction, adjusted pH value to 7 with CH₃COOD, after addition ofwater, the resulting mixture was extracted with EA (100 mL). Thecombined organic layer was washed with water and brine, dried overNa₂SO₄, and concentrated to give 7 (5.7 g) which was used for the nextstep. ESI-LCMS: m/z 401 [M+H]⁺.

Preparation of (8): To a solution of 7 (5.7 g) in pyridine (60 mL) wasadded BzCl (10.0 g, 71.3 mmol) under ice bath. The reaction mixture wasstirred at r.t. for 2.5 hrs. LCMS showed 7 was consumed. The mixture wasdiluted with EA and water was added. The product was extracted with EA.The crude was purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=7/3;Detector, UV 254 nm. This resulted in to give the crude 8 (6.2 g, 8.7mmol, 57% yield, over two steps) as a white solid. ESI-LCMS: m/z 713[M+H]⁺.

Preparation of (9): To a solution of 8 (6.2 g, 8.7 mmol) in pyridine (70mL) and was added 1M NaOH (MeOH/H₂O=4/1) (24 mL). LCMS showed 8 wasconsumed. The mixture was added saturated NH₄Cl till pH=7.5. The mixturewas diluted with water and EA. The organic layer was washed with brineand dried over Na₂SO₄ and concentrated to give the crude. The crude waspurified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=67/33Detector, UV 254 nm. This resulted in to give the product 10 (4.3 g, 8.5mmol, 98% yield) as a white solid. ESI-LCMS: m/z 505 [M+H]⁺; ¹H-NMR (400MHz, DMSO-d6): δ 11.23 (m, 1H), 8.77 (m, 2H), 8.06-8.04 (m, 2H),7.66-7.63 (m, 2H), 7.57-7.53 (m, 3H), 6.16-6.14 (d, J=8.0 Hz, 1H), 5.17(m, 1H), 4.60-4.52 (m, 2H), 3.34 (m, 1H), 0.93 (m, 9H), 0.14 (m, 6H).

Preparation of (10): To a stirred solution of 9 (4.3 g, 8.5 mmol) inpyridine (45 mL) were added DMTrCl (3.3 g, 9.8 mmol) at r.t. And thereaction mixture was stirred at r.t for 2.5 hr. With ice-bath cooling,the reaction was quenched with water and the product was extracted intoEA. The organic layer was washed with brine and dried over Na₂SO₄ andconcentrated to give the crude. The crude was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=97/3 Detector, UV 254 nm. Thisresulted in to give the product 10 (6.5 g, 8.1 mmol, 95% yield) as awhite solid. ESI-LCMS: m/z 807 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ11.23 (m, 1H), 8.70-8.68 (m, 2H), 8.04-8.02 (m, 2H), 7.66-7.62 (m, 1H),7.56-7.52 (m, 2H), 7.35-7.26 (m, 2H), 7.25-7.17 (m, 7H), 6.85-6.82 (m,4H), 6.18-6.16 (d, J=8.0 Hz, 1H), 4.73-4.70 (m, 1H), 4.61-4.58 (m, 1H),3.71 (m, 6H), 3.32 (m, 1H), 0.83 (m, 9H), 0.09-0.03 (m, 6H).

Preparation of (11): To a solution of 10 (3.5 g, 4.3 mmol) in THF (35mL) was added 1 M TBAF solution (5 mL). The reaction mixture was stirredat r.t. for 1.5 h. LCMS showed 10 was consumed completely. Water (100mL) was added. The product was extracted with EA (100 mL) and theorganic layer was washed with brine and dried over Na₂SO₄. Then theorganic layer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=62/38; Detector, UV 254 nm. Thisresulted in to give 11 (2.7 g, 3.9 mmol, 90.7%) as a white solid.ESI-LCMS: m/z 693 [M+H]⁺.

Preparation of Example 40 monomer: To a suspension of 11(2.7 g, 3.9mmol) in DCM (30 mL) was added DCI (0.39 g, 3.3 mmol) and CEP[N(iPr)₂]₂(1.4 g, 4.7 mmol). The mixture was stirred at r.t. for 2 h. LC-MS showed11 was consumed completely. The solution was washed with water twice andwashed with brine and dried over Na₂SO₄. Then concentrated to give aresidue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=73/27; Detector, UV 254 nm. This resulted in to give Example 40monomer (3.3 g, 3.7 mmol, 94.9%) as a white solid. ESI-LCMS: m/z 893[M+H]+; ¹H-NMR (400 MHz, DMSO-d6): δ=11.24 (m, 1H), 8.66-8.64 (m, 2H),8.06-8.03 (m, 2H), 7.65-7.53 (m, 3H), 7.42-7.38 (m, 2H), 7.37-7.34 (m,2H), 7.25-7.19 (m, 7H), 6.86-6.80 (m, 4H), 6.20-6.19 (d, J=4.0 Hz, 1H),4.78 (m, 2H), 4.22-4.21 (m, 1H), 3.92-3.83 (m, 1H), 3.72 (m, 6H),3.62-3.57 (m, 3H), 2.81-2.78 (m, 1H), 2.64-2.61 (m, 1H), 1.17-1.04 (m,12H); ³¹P-NMR (162 MHz, DMSO-d6): δ 149.51, 149.30.

Example 41. Synthesis of Monomer

Preparation of (3): To the solution of 1 (70 g, 138.9 mmol) in dryacetonitrile (700 mL) was added 2 (27.0 g, 166.7 mmol), BSA (112.8 g,555.5 mmol). The mixture was stirred at 50° C. for 1 h. Then the mixturewas cooled to −5° C. and TMSOTf (46.2 g, 208.3 mmol) slowly added to themixture. Then the reaction mixture was stirred at r.t for 48 h. Then thesolution was cooled to 0° C. and saturated aq. NaHCO₃ was added and theresulting mixture was extracted with EA. The combined organic layer waswashed with water and brine, dried over Na₂SO₄, and concentrated underreduced pressure to give a residue which was purified by silica gelcolumn chromatography (eluent, PE: EA=3:1-1:1) to give 3 (70 g, 115.3mmol, 81.6%) as a white solid. ESI-LCMS: m/z 605 [M−H]⁺.

Preparation of (4): To the solution of 3 (70.0 g, 115.3 mmol) inmethylammonium solution (1 M, 700 mL), and the reaction mixture wasstirred at 40° C. for 15 h. After completion of reaction, the resultingmixture was concentrated. The residue was crystallized from EA. Solidwas isolated by filtration, washed with PE and dried overnight at 45° C.in vacuum to give 4 (31.0 g, 105.4 mmol, 91.1%) as a white solid.ESI-LCMS: m/z 295 [M+H]⁺; ¹H-NMR (400 MHz, DMSO): δ 11.63 (s, 1H),8.07-7.99 (m, 1H),7.81 (d, J=8.4 Hz, 1H), 7.72-7.63 (m, 1H), 7.34-7.26(m, 1H), 6.18 (d, J=6.4 Hz, 1H), 5.24 (s, 1H), 5.00 (s, 2H), 4.58-4.47(m, 1H), 4.19-4.10 (m, 1H), 3.85-3.77 (m, 1H), 3.75-3.66 (m, 1H),3.66-3.57 (m, 1H).

Preparation of (5): To the solution of 4 (20.0 g, 68.0 mmol) in dry DMF(200 mL) was added DPC (18.9 g, 88.0 mmol) and NaHCO₃(343 mg, 4 mmol) atr.t, and the reaction mixture was stirred at 150° C. for 35 min. Aftercompletion of reaction, the resulting mixture was poured into tert-Butylmethyl ether (4 L). Solid was isolated by filtration, washed with PE anddried in vacuum to give crude 5 (21.0 g) as a brown solid which was useddirectly for next step (ref for 5, Journal of Organic Chemistry, 1989,vol. 33, p. 1219-1225). ESI-LCMS: m/z 275 [M−H]⁻.

Preparation of (6): To the solution of 5 (crude, 21.0 g) in Pyridine(200 mL) was added AgNO₃ (31.0 g, 180.0 mmol) and collidine (88.0 g, 720mmol) and TrtCl (41.5 g, 181 mmol) at r.t, and the reaction mixture wasstirred at r.t for 15 h. After addition of water, the resulting mixturewas extracted with EA. The combined organic layer was washed with waterand brine, dried over Na₂SO₄, and concentrated to give the crude. Thecrude was by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give 6 (10.0 g, 13.1 mmol, 20%yield over 3 steps) as a white solid. ESI-LCMS: m/z 761 [M+H]⁺.

Preparation of (7): To the solution of 6 (10.0 g, 13.1 mmol) in THF (100mL) was added 6 N NaOH (30 mL) at r.t, and the reaction mixture wasstirred at r.t for 1 hr. After addition of NH₄Cl, the resulting mixturewas extracted with EA. The combined organic layer was washed with waterand brine, dried over Na₂SO₄, and concentrated under reduced pressureand the residue was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=9/1; Detector, UV 254 nm. This resulted in to give 7 (9.3 g,11.9 mmol, 90%) as a white solid. ESI-LCMS: m/z 777 [M−H]⁻;¹H-NMR (400MHz, DMSO-d6): δ 11.57 (s, 1H), 8.02 (d, J=8.7 Hz, 1H), 7.88-7.81 (m,1H), 7.39-7.18 (m, 30H), 7.09-6.99 (m, 30H), 6.92-6.84 (m, 30H), 6.44(d, J=4.0 Hz, 1H), 4.87 (d, J=4.0 Hz, 1H), 4.37-4.29 (m, 1H), 4.00-3.96(m, 1H), 3.76-3.70 (m, 1H), 3.22-3.13 (m, 1H), 3.13-3.04 (m, 1H).

Preparation of (8): To the solution of 7 (8.3 g, 10.7 mmol) in dry DCM(80 mL) was added Pyridine (5.0 g, 64.2 mmol) and DAST (6.9 g, 42.8mmol) at 0° C., and the reaction mixture was stirred at r.t for 15 hr.After addition of NH₄Cl, the resulting mixture was extracted with DCM.The combined organic layer was washed with water and brine, dried overNa₂SO₄, and concentrated under reduced pressure and the residue waspurified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give 8 (6.8 g, 8.7 mmol, 81.2%)as a white solid. ESI-LCMS: m/z 779 [M−H]⁺; ¹⁹F-NMR (376 MHz, DMSO-d6):δ −183.05.

Preparation of (9): To the solution of 8 (5.8 g, 7.5 mmol) in dry ACN(60 mL) was added TEA (1.5 g, 15.1 mmol), DMAP (1.84 g, 15.1 mmol) andTPSCI (4.1 g, 13.6 mmol) at r.t, and the reaction mixture was stirred atroom temperature for 3 h under N₂ atmosphere. After completion ofreaction, the mixture was added NH₃.H₂O (12 mL). After addition ofwater, the resulting mixture was extracted with EA. The combined organiclayer was washed with water and brine, dried over Na₂SO₄, andconcentrated under reduced pressure and the residue was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give 9 (5.5 g, 7 mmol, 90.2%) as a white solid. ESI-LCMS:m/z 780 [M+H]⁺.

Preparation of (10): To a solution of 9 (5.5 g, 7 mmol) in DCM (50 mL)with an inert atmosphere of nitrogen was added pyridine (5.6 g, 70.0mmol) and BzCl (1.2 g, 8.5 mmol) in order at 0° C. The reaction solutionwas stirred for 30 minutes at room temperature. The solution was dilutedwith DCM (100 mL) and the combined organic layer was washed with waterand brine, dried over Na₂SO₄, and concentrated under reduced pressure togive a residue which was purified by silica gel column chromatography(eluent, PE: EA=5:1-2:1) to give 10 (5.4 g, 6.1 mmol, 90.6%) as a whitesolid. ESI-LCMS: m/z 884 [M+H]⁺; ¹⁹F-NMR (376 MHz, DMSO-d6): δ −183.64.

Preparation of (11): To the solution of 10 (5.4 g, 6.1 mmol) in thesolution of DCA (6%) in DCM (60 mL) was added TES (15 mL) at r.t, andthe reaction mixture was stirred at room temperature for 5-10 min. Aftercompletion of reaction, the resulting mixture was added NaHCO₃, theresulting mixture was extracted with DCM. The combined organic layer waswashed with water and brine, dried over Na₂SO₄, and concentrated underreduced pressure and the residue was crystallized from EA. Solid wasisolated by filtration, washed with PE and dried overnight at 450 invacuum to give 11(2.0 g, 5.0 mmol, 83.2%) as a white solid. ESI-LCMS:m/z 400 [M+H]⁺.

Preparation of (12): To a solution of 11 (2.0 g, 5.0 mmol) in dryPyridine (20 mL) was added DMTrCl (2.0 g, 6.0 mmol). The reactionmixture was stirred at r.t. for 2.5 h. LCMS showed 11 was consumed andwater (200 mL) was added. The product was extracted with EA (200 mL) andthe organic layer was washed with brine and dried over Na₂SO₄ andconcentrated to give the crude. The crude was purified by c.c. (PE:EA=4:1-1:1) to give crude 12. The crude was further purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give 12 (2.1 g, 3 mmol, 60%) as a white solid. ESI-LCMS:m/z 702 [M+H]⁺:H-NMR (400 MHz, DMSO-d6): δ 12.63 (s, 1H), 8.54 (d, J=7.8Hz, 1H), 8.25 (d, J=7.2 Hz, 2H), 7.82 (d, J=3.6 Hz, 2H), 7.67-7.58 (m,1H), 7.57-7.49 (m, 2H), 7.49-7.39 (m, 1H), 7.39-7.31 (m, 2H), 7.27-7.09(m, 7H), 6.82-6.69 (m, 4H), 6.23 (d, J=26.1 Hz, 1H), 5.59-5.49 (m, 1H),4.83-4.61 (m, 1H), 4.15-4.01 (m, 1H), 3.74-3.59 (m, 6H), 3.33-3.28 (m,1H), 3.16-3.05 (m, 1H). ¹⁹F-NMR (376 MHz, DMSO-d6): δ −191.66.

Preparation of Example 41 monomer: To a suspension of 12 (2.1 g, 3.0mmol) in DCM (20 mL) was added DCI (310 mg, 2.6 mmol) and CEP[N(iPr)₂]₂(1.1 g, 3.7 mmol). The mixture was stirred at r.t. for 1 h. LC-MS showed12 was consumed completely. The solution was washed with water twice andwashed with brine and dried over Na₂SO₄. Then concentrated to give thecrude. The crude was by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give Example 41 monomer (2.1 g,2.3 mmol, 80.0%) as a white solid. ESI-LCMS: m/z 902 [M+H]⁺; ¹H-NMR (400MHz, DMSO-d6): δ 12.64 (s, 1H), 8.54 (d, J=7.6 Hz, 1H), 8.24 (d, J=7.7Hz, 2H), 7.93-7.88 (m, 2H), 7.67-7.58 (m, 1H), 7.56-7.42 (m, 3H),7.41-7.29 (m, 2H), 7.27-7.08 (m, 7H), 6.82-6.64 (m, 4H), 6.37-6.18 (m,1H), 6.03-5.72 (m, 1H), 5.26-4.83 (m, 1H), 4.28-4.12 (m, 1H), 3.88-3.72(m, 1H), 3.71-3.37 (m, 9H), 3.15-3.00 (m, 1H), 2.83-2.75 (m, 1H),2.66-2.57 (m, 1H), 1.21-0.88 (m, 12H). ¹⁹F-NMR (376 MHz, DMSO-d6): δ−189.71. ³¹P-NMR (162 MHz, DMSO-d6): δ 149.48, 149.50, 148.95, 148.88.

Example 42. Synthesis of Monomer

Preparation of (3): To a solution of 1(40.0 g, 79.3 mmol), 1a (7.6 g,80.1 mmol) in ACN (100 mL). Then added BSA (35.2 g, 174.4 mmol) under N₂atmosphere. The mixture was stirred at 50° C. for 1 h until the solutionwas clear. Then cool down to 0° C. and dropped TMSOTf (18.5 g, 83.2mmol).The mixture was stirred at 75° C. for 1 h, TLC showed 1 wasconsumed completely. Then the solution was diluted with EA, washed withH₂O twice. The solvent was concentrated under reduced pressure and theresidue was used for next step. ESI-LCMS: m/z 540 [M+H]+.

Preparation of (3): To a solution of 2 (37.1 g, 68.7 mmol) in30%CH₂NH₂/MeOH solution (200 mL). The mixture was stirred at 25° C. for2 h. TLC showed 2 was consumed completely. The solvent was concentratedunder reduced pressure and the residue was washed with EA twice to give3 (12.5 g, 55.2 mmol) ( ref. for intermediate 3 Bioorganic & MedicinalChemistry Letters, 1996, Vol. 6, No. 4, pp. 373-378,) which was useddirectly for the next step. ESI-LCMS: m/z 228 [M+H]⁺.

Preparation of (4): To a solution of 3 (12.5 g, 55.2 mmol) in pyridine(125 mL) and added DMAP (1.3 g, 11.0 mmol), TrtCl (30.7 g, 110.5 mmol).The mixture was stirred at r.t. for 24 h. TLC showed 3 was consumedcompletely. H₂O was added to the mixture. Then filtered and the solutiondiluted with EA. The organic layer was washed with NaHCO₃ and brine. Thesolvent was concentrated under reduced pressure and then added ACN,filtered to give 4a (17.0 g, 35.4 mmol, 64% yield) as a white solid.

To a solution of 4a (17.0 g, 35.4 mmol) in DMF (200 mL), collidine (5.2g, 43.5 mmol), TrCl (13.1 g, 47.1 mmol) were added after 2 h and thenagain after 3 h TrCl (13.1 g, 47.1 mmol), AgNO₃ (8.0 g, 47.1 mmol). Themixture was stirred at 25° C. for 24 h. TLC showed 4a was consumedcompletely. Then filtered and the solution diluted with EA. The organiclayer was washed with NaHCO₃ and brine. The solvent was concentratedunder reduced pressure and then added ACN, filtered to get 4 (14.2 g,19.5 mmol, 54% yield) as a white solid. ESI-LCMS: m/z 712 [M+H]⁺;H-NMR(400 MHz, DMSO-d6): δ 7.83 (d, J=8 Hz, 2H), 7.42-7.20 (m, 30H), 6.18 (d,J=7 Hz, 1H), 6.09 (d, J=8 Hz, 2H), 5.60 (d, J=7 Hz, 1H), 4.22 (m, 1H),3.90 (d, J=5 Hz, 1H), 2.85 (d, J=10 Hz, 1H), 2.76 (s, 1H), 2.55-2.50(dd, 1H).

Preparation of (5): To a solution of 4 (14.2 g, 19.9 mmol) in DCM (150mL), DMAP (2.4 g, 19.9 mmol), TEA (4.0 g, 39.9 mmol, 5.6 mL) were added.Then cool down to 0° C., TfCl (6.7 g, 39.9 mmol) dissolved in DCM (150mL) were dropped. The mixture was stirred at 25° C. for 1 h. TLC showed4 was consumed completely. Then filtered and the solution diluted withEA. The organic layer was washed with NaHCO₃ and brine. The solvent wasconcentrated under reduced pressure to get 5 (16.8 g, 19.9 mmol) as abrown solid. ESI-LCMS: m/z 844 [M+H]⁺.

Preparation of (6): To a solution of 5 (16.8 g, 19.9 mmol) in DMF (200mL), KOAc (9.7 g, 99.6 mmol) were added, The mixture was stirred at 25°C. for 14 h and 50° C. for 3 h, TLC showed 5 was consumed completely.Then filtered and the solution diluted with EA. The organic layer waswashed with H₂O and brine. The solvent was concentrated under reducedpressure to get 6a (15.0 g, 18.9 mmol, 90% yield) as a brown solid. To asolution of 6a (15.0 g, 19.9 mmol) in 30% CH₃NH₂/MeOH solution (100 mL)were added. The mixture was stirred at 25° C. for 2 h, TLC showed 6a wasconsumed completely. Then the solvent was concentrated under reducedpressure and the residue was purified by cc (0-5% MeOH in DCM) to give 6(11.6 g, 16.3 mmol, 82% yield) as a yellow solid. ESI-LCMS: m/z 712[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 7.59 (d, J=8 Hz, 2H), 7.37-7.22 (m,30H), 6.01 (d, J=8 Hz, 2H), 5.84 (d, J=3 Hz, 1H), 5.42 (d, J=4 Hz, 1H),3.78-3.70 (m, 3H), 3.10 (t, J=9 Hz, 1H), 2.53 (d, J=4 Hz, 6H), 1.77 (s,6H).

Preparation of (7): To a solution of 6 (11.6 g, 16.32 mmol) in DCM (200mL), DAST (7.9 g, 48.9 mmol)were added at 0° C., The mixture was stirredat 25° C. for 16 h, TLC showed 6 was consumed completely. Then thesolution was diluted with EA, washed with NaHCO₃ twice, The solvent wasconcentrated under reduced pressure the residue purified byFlash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=4/1; Detector, UV 254 nm. Thisresulted in to give 7 (11.6 g, 13.8 mmol, 84% yield) as a white solid.ESI-LCMS: m/z 714 [M+H]⁺.

Preparation of (8): To a solution of 7 (11.6 g, 16.2 mmol) in DCM (100mL) was added TFA (10 mL). The mixture was stirred at 20° C. for 1 h.TLC showed 7 was consumed completely. Then the solution was concentratedunder reduced pressure the residue was purified by silica gel column(0˜20% MeOH in DCM) and Flash-Prep-HPLC with the followingconditions(IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=0/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=1/3within 25 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=0/1; Detector, UV 254 nm. This resulted in to give 9 (1.7 g,7.2 mmol, 45% yield) as a white solid. ESI-LCMS: m/z 229.9 [M+H]⁺;¹H-NMR (400 MHz, DMSO-d6): δ 7.91 (d, J=8 Hz, 2H), 6.14 (d, J=8 Hz, 2H),5.81-5.76 (m, 2H), 5.28 (t, J=5 Hz, 1H), 5.13-4.97 (t, J=4 Hz, 1H), 4.23(m, 1H), 3.97 (m, 1H), 3.74-3.58 (m, 2H); ¹⁹F-NMR (376 MHz, DMSO-d6): δ−206.09.

Preparation of (9): To a solution of 8 (1.4 g, 6.1 mmol) in pyridine (14mL) was added DMTrC1 (2.5 g, 7.3 mmol) at 20° C. The mixture was stirredat 20° C. for 1 h. TLC showed 8 was consumed completely. Water was addedto the reaction. The product was extracted with EA, The organic layerwas washed with NaHCO₃ and brine. Then the solution was concentratedunder reduced pressure and the residue was purified by Flash-Prep-HPLCwith the following conditions (IntelFlash-1): Column, C18 silica gel;mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/3 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=4/1 within 25 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=1/1; Detector, UV 254 nm. This resulted in togive 9 (2.5 g, 4.6 mmol, 76 yield) as a white solid. ESI-LCMS: m/z 532.2[M+H]:H-NMR (400 MHz, DMSO-d6): δ 7.87-7.84 (m, 2H), 7.40-7.22 (m, 9H),6.91-6.87 (m, 4H), 5.98-5.95 (m, 2H), 5.88-5.77 (m, 2H), 5.16-5.02 (m,1H), 4.42 (m, 1H), 4.05 (m, 1H), 3.74 (s, 6H), 3.35 (m, 2H); ¹⁹F-NMR(376 MHz, DMSO-d6): δ −202.32.

Preparation of Example 42 monomer: To a solution of 9 (2.2 g, 4.1 mmol)in DCM (20 mL) was added DCI (415 mg, 3.5 mmol) and CEP (1.5 g, 4.9mmol) under N₂ pro. The mixture was stirred at 20° C. for 0.5 h. TLCshowed 9 was consumed completely. The product was extracted with DCM,The organic layer was washed with H₂O and brine. Then the solution wasconcentrated under reduced pressure and the residue was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. Thisresulted in to give Example 42 monomer (2.6 g, 3.5 mmol, 85% yield) as awhite solid. ESI-LCMS: m/z 732.2 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ7.87-7.84 (m, 2H), 7.40-7.22 (m, 9H), 6.91-6.87 (m, 4H), 5.98-5.95 (m,2H), 5.90-5.88 (m, 1H), 5.30-5.17 (m, 1H), 4.62 (m, 1H), 4.19 (m, 1H),3.78-3.73 (m, 7H), 3.62-3.35 (m, 5H), 2.78 (t, J=5 Hz, 1H), 2.63 (t, J=6Hz, 1H), 1.14-0.96 (m, 12H); ¹⁹F-NMR (376 MHz, DMSO-d6): δ −200.77,200.80, 201.62, 201.64. ³¹P-NMR (162 MHz, DMSO-d6): δ 150.31, 150.24,149.66, 149.60.

Example 43. Synthesis of End Cap Monomer

Preparation of (8): To a stirred solution of 7 (13.4 g, 35.5 mmol,Scheme 5) in DMSO (135 mL) were added EDCI (6.3 g, 32.9 mmol) andpyridine (0.9 g, 10.9 mmol), TFA (0.6 g, 5.5 mmol) at r.t. And thereaction mixture was stirred at r.t for 2 h. LCMS showed 7 consumedcompletely. The reaction was quenched with water and the product wasextracted with EA (1800 mL). The organic phase was washed by brine,dried over Na₂SO₄, The organic phase was evaporated to dryness underreduced pressure to give a residue 8 (13.2 g, 35.3 mmol, 99.3% yield).Which was used directly to next step. ESI-LCMS: m/z=375 [M+H₂O]⁺

Preparation of (10): A solution of 8 (13.2 g, 35.3 mmol), 9 (26.8 g,42.3 mmol, Scheme 18) and K₂CO₃ (19.5 g, 141.0 mmol) in dry THF (160 mL)and D20 (53 mL) was stirred at r.t. 17 h. LCMS showed most of 8 wasconsumed. The product was extracted with EA (2500 mL) and the organiclayer was washed with brine and dried over Na₂SO₄. Then the organiclayer was concentrated to give a residue which was purified by c.c. (PE:EA=10:1-1:2) to give product 10 (8.1 g, 11.8 mmol, 33.4% yield) as awhite solid. ESI-LCMS m/z=682 [M+H]⁺, ¹H-NMR (400 MHz, DMSO-d6): δ 11.42(s, 1H), 7.69-7.71 (d, J=8.1 Hz, 1H), 5.78-5.79 (d, J=3.7 Hz, 1H),5.65-5.67 (m, 1H), 5.59-5.63 (m, 4H), 4.29-4.35 (m, 2H), 3.97-3.99 (m,1H), 1.15 (s, 18H), 0.87 (s, 9H), 0.07-0.08 (d,.J=5.1 Hz, 6H). ³¹P-NMR(162 MHz, DMS 046) S 16.62.

Preparation of (11): To a round-bottom flask was added 10 (7.7 g, 11.1mmol) in a mixture of HCOOH (80 mL) and H₂O (80 mL). The reactionmixture was stirred at 40° C. for 3 h. LCMS showed the 10 was consumedcompletely. The reaction mixture was adjusted the pH=7.0 withcon.NH₃.H₂O (100 mL). Then the mixture was extracted with DCM (100mL*3). The combined DCM layer was dried over Na₂SO₄. Filtered andfiltrate was concentrated to give crude which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/2 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O; Detector, UV 254 nm. To giveproduct 11 (5.5 g, 9.6 mmol, 86.1% yield) as a white solid. ESI-LCMSm/z=568 [M+H]⁺, ¹H-NMR (400 MHz, DMSO-d6): δ 11.42 (s, 1H, exchangedwith D20), 7.62-7.64 (d, J=8.1, 1H), 5.81-5.82 (d, J=4.3, 1H), 5.58-5.66(m, 5H), 5.52-5.53 (d, J=6.6, 1H), 4.34-4.37 (m, 1H), 4.09-4.13 (m, 1H),3.94-3.96 (t, J=9.7, 1H), 1.15 (s, 18H), 0 (s, 1H). ³¹P NMR (162 MHz,DMSO-d6) δ 17.16.

Preparation of Example 43 monomer: To a solution of 11 (5.3 g, 9.3 mmol)in DCM (40 mL) was added the DCI (1.1 g, 7.9 mmol), then CEP[N(ipr)₂]₂(3.4 g, 11.2 mmol) was added. The mixture was stirred at r.t. for 1 h.LCMS showed 11 consumed completely. The reaction mixture was washed withH₂O (50 mL*2) and brine (50 mL*1). Dried over Na₂SO₄ and concentrated togive crude which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. The product was concentrated to giveExample 43 monomer (6.2 g, 8.0 mmol, 85.6% yield) as a white solid.ESI-LCMS m/z=768 [M+H]⁺;H-NMR (400 MHz, DMSO-d6): δ 11.43 (s, 1H),7.68-7.71 (m, 1H), 5.79-5.81 (m, 1H), 5.58-5.67 (m, 5H), 4.34-4.56 (m,2H), 4.14-4.17 (m, 1H), 3.54-3.85 (m, 4H), 2.78-2.81 (m, 2H), 1.13-1.17(m, 30H). ³¹P-NMR (162 MHz, DMSO-d6): δ 149.66, 149.16, 16.84, 16.56.

Example 44. Synthesis of Monomer

Preparation of (2): To a solution of 1(20.0 g, 66.4 mmol) in dry DMF(400 mL) was added sodium hydride (1.9 g, 79.7 mmol) for 30 min, thenwas added CD3I (9.1 g, 79.7 mmol) in dry DCM (40 mL) at −20° C. for 5.5hr. LCMS showed the reaction was consumed. The mixture was filtered andthe clear solution was evaporated to dryness and was evaporated withCH₃₀H. The crude was purified by silica gel column (SiO₂,DCM/MeOH=50:1-10:1). This resulted in to give the product 2 (7.5 g, 23.5mmol, 35.5% yield) as a solid. ESI-LCMS: m/z 319 [M+H]⁺; H-NMR (400 MHz,DMSO-d3): δ=8.38 (m, 1H), 6.97 (m, 2H), 5.93-5.81 (m, 1H), 5.27-5.26 (d,J=4 Hz, 1H), 5.13-5.11 (m, 1H), 4.39-4.31 (m, 1H), 4.31-4.25 (m, 1H),3.96-3.94 (m, 1H), 3.66-3.63 (m, 1H), 3.63-3.56 (m, 1H).

Preparation of (3): To a solution of 2 (7.5 g, 23.5 mmol) in dry DMF (75mL) was added Imidazole (5.6 g, 82.3 mmol) and TBSCl (8.9 g, 58.8 mmol).The mixture was stirred at r.t. over night. LCMS showed 2 was consumedcompletely. The reaction was quenched with water (300 mL). The productwas extracted into ethyl acetate (100 mL). The organic layer was washedwith brine and dried over anhydrous Na₂SO₄. The solvent was removed togive the cured 3 (9.8 g) as a solid witch used for the next step.ESI-LCMS: m/z 547 [M+H]⁺.

Preparation of (4): To a solution of 3 (9.8 g) in THF (40 mL) was addedTFA (10 mL) and water (10 mL) at 0° C. The reaction mixture was stirredat 0° C. for 5 h. LC-MS showed 3 was consumed completely. Con. NH₄OH wasadded to the mixture at 0° C. to quench the reaction until the pH=7.5.The product was extracted into ethyl acetate (200 mL). The organic layerwas washed with brine and dried over anhydrous Na₂SO₄. The solvent wasremoved to give the cured 4 (8.4 g) as a solid witch used for the nextstep. ESI-LCMS: m/z 433 [M+H]⁺.

Preparation of (5): To a solution of 4 (8.4 g) in DCM/H₂O=2:1 (84 mL)was added DAIB (18.8 g, 58.4 mmol) and TEMPO (0.87 g, 5.8 mmol). Thereaction mixture was stirred at 40° C. for 2 h. LCMS showed 4 wasconsumed. The mixture was diluted with DCM and water was added. Theproduct was extracted with DCM. The organic layer was washed with brineand dried over anhydrous Na₂SO₄. The solution was then concentratedunder reduced pressure. This resulted in to give 5 (14.4 g) as a whitesolid. ESI-LCMS: m/z 447 [M+H]⁺.

Preparation of (6): To a solution of 5 (14.4 g) in toluene (90 mL) andmethanol (60 mL) was added 2M TMSCHN₂ (8.9 g, 78.1 mmol) till the yellowcolor not disappear at r.t. for 10 min. LCMS showed 5 was consumed. Thecrude was purified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=65/35Detector, UV 254 nm. This resulted in to give the product 6 (3.5 g, 7.6mmol, 32.3% yield over three steps, 70% purity) as a white solid.ESI-LCMS: m/z 461 [M+H]⁺.

Preparation of (7): To the solution of 6 (3.5 g, 7.6 mmol) in dryTHF/MeOD/D20=10/2/1 (45 mL) was added NaBD₄ (0.96 g, 22.8 mmol). And thereaction mixture was stirred at r.t for 2.5 hr. After completion ofreaction, the resulting mixture was added CH₃COOD to pH=7, afteraddition of water, the resulting mixture was extracted with EA (100 mL).The combined organic layer was washed with water and brine, dried overNa₂SO₄, and concentrated to give 7 (3.3 g) which was used for the nextstep. ESI-LCMS: m/z 435 [M+H]⁺.

Preparation of (8): To a solution of 7 (3.3 g) in dry DCM (30 mL) wasadded pyridine (5.9 g, 74.5 mmol) and iBuCl (2.4 g, 22.4 mmol) in DCM (6mL) under ice bath. The reaction mixture was stirred at 0° C. for 2.5hr. LCMS showed 7 was consumed. The mixture was diluted with EA andwater was added. The product was extracted with EA. The crude waspurified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=87/13;Detector, UV 254 nm. This resulted in to give the crude 8 (1.6 g, 2.8mmol, 36.8% yield over two steps) as a white solid. ESI-LCMS: m/z 575[M+H]⁺.

Preparation of (9): To a solution of 8 (1.6 g, 2.8 mmol,) inH₂O/dioxane=1:1 (30 ml) was added K₂CO₃ (772.8 mg, 5.6 mmol) and DABCO(739.2 mg, 2.9 mmol). The reaction mixture was stirred at 50° C. for 3hr. LCMS showed 8 was consumed. The mixture was diluted with EA andwater was added. The product was extracted with EA. The combined organiclayer was washed with water and brine, dried over Na₂SO₄, andconcentrated to give 9 (1.8 g) which was used for the next step.ESI-LCMS: m/z 557 [M+H]⁺.

Preparation of (10): To a solution of 9 (1.8 g) in pyridine (20 mL) andwas added 2M NaOH (MeOH/H₂O=4/1) (5 mL) at 0° C. for 1 h. LCMS showed 9was consumed. The mixture was added saturated NH₄Cl till pH=7.5. Themixture was diluted with water and EA. The organic layer was washed withbrine and dried over Na₂SO₄ and concentrated to give the crude. Thisresulted in to give the product 10 (1.5 g) as a white solid which wasused for the next step. ESI-LCMS: m/z 487 [M+H]⁺.

Preparation of (11): To a stirred solution of 10 (1.5 g) in pyridine (20mL) were added DMTrCl (1.1 g, 3 mmol) at r.t. And the reaction mixturewas stirred at r.t for 2.5 hr. With ice-bath cooling, the reaction wasquenched with water and the product was extracted into EA. The organiclayer was washed with brine and dried over Na₂SO₄ and concentrated togive the crude. The crude was purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 25 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=7/3 Detector, UV 254 nm. This resulted in togive the product 11(1.9 g, 2.4 mmol, 85.7% yield over two steps) as awhite solid. ESI-LCMS: m/z 789.3 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ12.10 (m, 1H), 11.63 (m, 1H), 8.20 (m, 1H), 7.35-7.33 (m, 2H), 7.29-7.19(m, 7H), 6.86-6.83 (m, 4H), 5.89-5.88 (d, J=4 Hz, 1H), 4.40-4.28 (m,2H), 3.72 (m, 6H), 2.81-2.76 (m, 1H), 1.13-1.11 (m, 6H), 0.80 (m, 9H),0.05-0.01 (m, 7H).

Preparation of (12): To a solution of 11 (1.9 g, 2.4 mmol) in THF (20mL) was added 1 M TBAF solution (3 mL). The reaction mixture was stirredat r.t. for 1.5 h. LCMS showed 11 was consumed completely. Water (100mL) was added. The product was extracted with EA (50 mL) and the organiclayer was washed with brine and dried over Na₂SO₄. Then the organiclayer was concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=58/42; Detector, UV 254 nm. Thisresulted in to give 12 (1.5 g, 2.2 mmol, 91.6% yield) as a white solid.ESI-LCMS: m/z 675.3 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): S 12.09 (m, 1H),11.60 (m, 1H), 8.14 (m, 1H), 7.35-7.27 (m, 2H), 7.25-7.20 (m, 7H),6.85-6.80 (m, 4H), 5.96-5.94 (d, J=8 Hz, 1H), 5.26-5.24 (m, 1H),4.35-4.28 (m, 2H), 3.72 (m, 6H), 3.32 (m, 1H), 2.79-2.72 (m, 1H),1.13-1.11 (m, 6H).

Preparation of Example 44 monomer: To a suspension of 11(1.5 g, 2.2mmol) in DCM (15 mL) was added DCI (220.8 mg, 1.9 mmol) andCEP[N(iPr)₂]₂ (795.7 mg, 2.6 mmol) under N₂ pro. The mixture was stirredat r.t. for 2 h. LCMS showed 11 was consumed completely. The solutionwas washed with water twice and washed with brine and dried over Na₂SO₄.Then concentrated to give a residue which was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 20 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=4/1; Detector, UV 254 nm. Thisresulted in to give Example 44 monomer (1.6 g, 1.8 mmol, 83% yield) as awhite solid. ESI-LCMS: m/z 875 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ12.12 (m, 1H), 11.60 (m, 1H), 8.15 (m, 1H), 7.37-7.29 (m, 2H), 7.27-7.20(m, 7H), 6.86-6.81 (m, 4H), 5.94-5.88 (m, 1H), 4.54-4.51 (m, 2H),4.21-4.20 (m, 1H), 3.73-3.54 (m, 10H), 2.80-2.75 (m, 1H), 2.61-2.58 (m,1H), 1.19-1.11 (m, 19H). ³¹P-NMR (162 MHz, DMSO-d6): δ=149.77, 149.71.

Example 45. Synthesis of Monomer

Preparation of (2): To a solution of 1(50.0 g, 99.2 mmol) and 1a (11.3g, 119.0 mmol) in ACN (500.0 mL). Then added BSA (53.2 g, 218.0 mmol)under N₂ Pro. The mixture was stirred at 50° C. for 1 h until thesolution was clear. Then cool down to 0° C. and dropped TMSOTf (26.4 g,119.0 mmol).The mixture was stirred at 75° C. for 1 h, TLC showed 1 wasconsumed completely. The reaction was quenched by sodium bicarbonatesolution at 0° C., then the solution was diluted with EA, washed withH₂O twice. The solvent was concentrated under reduced pressure and thecrude 2 (60.1 g) was used for next step. ESI-LCMS: m/z 540.2 [M+H]⁺.

Preparation of (3): To a solution of 2 (60.1 g) in CH₃NH₂/ethanol (500.0mL). The mixture was stirred at 25° C. for 2 h. TLC showed 2 wasconsumed completely. The solvent was concentrated under reduced pressureand the residue was purified by c.c. (MeOH:DCM=50:1-10:1) to give 3(22.0 g, 96.9 mmol, 97.3% yield over two steps). ESI-LCMS: m/z 228.0[M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 8.01-7.98 (m, 1H), 7.43-7.38 (m,1H), 6.37-6.35 (m, 1H), 6.27-6.23 (m, 1H), 6.03 (d, J=3.5 Hz, 1H), 5.39(d, J=4.2 Hz, 1H), 5.11 (t, J=5.1 Hz, 1H), 5.03 (d, J=5.1 Hz, 1H),3.98-3.95 (m, 2H), 3.91-3.88 (m, 1H), 3.74-3.57 (m, 2H).

Preparation of (4): To a solution of 3 (22.0 g, 96.9 mmol) in pyridine(250.0 mL), TrtCl (30.7 g, 110.5 mmol) was added. The mixture wasstirred at 25° C. for 24 h. TLC showed 3 was consumed completely, H₂Owas added to the mixture. Then filtered and the filtrate diluted withEA, the organic layer was washed with NaHCO₃ and brine. The solvent wasconcentrated under reduced pressure and then purified by c.c.(PE/EA=5:1-0:1) to give 4 (38.8 g, 82.5 mmol, 85.1% yield) as a whitesolid. ESI-LCMS: m/z 470.1 [M+H]⁺.

Preparation of (5): To a solution of 4 (38.8 g, 82.5 mmol) in DMF (500.0mL), collidine (10.0 g, 107.3 mmol), TrtCl (27.6 g, 99.1 mmol) wereadded followed by AgNO₃ (18.0 g, 105.1 mmol). The mixture was stirred at25° C. for 4 h. TLC showed 4 was consumed completely. Then filtered andthe filtrate diluted with EA. The organic layer was washed with NaHCO₃and brine. The solvent was concentrated under reduced pressure and thenpurified by c.c. (PE/EA=5:1-1:1) to give a mixture of 5 (52.3 g, 73.5mmol, 86.3% yield) as white solid. ESI-LCMS: m/z 711.1 [M+H]⁺.

Preparation of (6): To a solution of 5 (52.3 g, 73.5 mmol) in DCM (500.0mL), DMAP (8.9 g, 73.5 mmol), TEA (14.9 g, 147.3 mmol, 20.6 mL) wereadded, cool down to 0° C., TfCl (16.1 g, 95.6 mmol) dissolved in DCM(100.0 mL) were dropped. The mixture was stirred at 25° C. for 1 h. TLCshowed 5 was consumed completely. Then filtered and the solution dilutedwith EA. The organic layer was washed with NaHCO₃ and brine. The solventwas concentrated under reduced pressure to get crude 6 (60.2 g) as abrown solid. ESI-LCMS: m/z 844.2 [M+H]⁺.

Preparation of (7): To a solution of 6 (60.2 g) in DMF (500.0 mL), KOAc(36.1 g, 367.8 mmol) were added, The mixture was stirred at 25° C. for14 h and 50° C. for 3 h, TLC showed 6 was consumed completely. Thenfiltered and the solution diluted with EA. The organic layer was washedwith H₂O and brine. The solvent was concentrated under reduced pressure,residue was purified by c.c. (PE/EA=5:1-1:1) to give 7 (28.0 g, 39.3mmol, 53.5% yield) as yellow solid. ESI-LCMS: m/z 710.2 [M−H]⁻; H-NMR(400 MHz, DMSO-d6): δ 7.37-7.25 (m, 33H), 6.34-6.31 (m, 2H), 6.13-6.10(m, 1H), 5.08 (d, J=4.2 Hz, 1H), 3.99 (d, J=7.6 Hz, 1H), 3.74 (s, 1H),3.12 (t, J=9.2 Hz, 1H), 2.72-2.69 (m, 1H).

Preparation of (8): To a solution of 7 (28.0 g, 39.3 mmol) in DCM (300.0mL), DAST (31.6 g, 196.6 mmol) was added at 0° C., the mixture wasstirred at 25° C. for 16 h, TLC showed 7 was consumed completely. Thenthe solution was diluted with EA, washed with NaHCO₃ twice, the solventwas removed under reduced pressure, residue was purified by c.c.(PE/EA=5:1-3:1) to give 8 (5.0 g, 7.0 mmol, 17.8% yield) as a whitesolid. ESI-LCMS: m/z 748.2 [M+2NH₄]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ7.57-7.18 (m, 35H), 6.30 (d, J=8.8 Hz, 1H), 6.00 (d, J=19.5 Hz, 1H),5.92-5.88 (m, 1H), 4.22-4.17 (m, 2H), 3.94 (s, 0.5H), 3.80 (s, 0.5H),3.35-3.31 (m, 1H), 3.14-3.10 (m, 1H); ¹⁹F-NMR (376 MHz, DMSO-d6): δ−193.54.

Preparation of (9): To a solution of 8 (5.0 g, 7.0 mmol) in DCM (60.0mL) was added DCA (3.6 mL) and TES (15.0 mL). The mixture was stirred at20° C. for 1 h, TLC showed 8 was consumed completely. Then the solutionwas concentrated under reduced pressure, the residue was purified byFlash-Prep-HPLC with the following conditions (IntelFlash-1): Column,C18 silica gel; mobile phase, CH₃CN/H₂O (0.5% NH₄HCO₃)=0/1 increasing toCH₃CN/H₂O (0.5% NH₄HCO₃)=1/3 within 25 min, the eluted product wascollected at CH₃CN/H₂O (0.5% NH₄HCO₃)=0/1; Detector, UV 254 nm. Thisresulted in to give 9 (1.6 g, 6.9 mmol, 98.5% yield) as a white solid.ESI-LCMS: m/z 229.9 [M+H]⁺; ¹H-NMR (400 MHz, DMSO-d6): δ 8.06-8.04 (m,1H), 7.48-7.43 (m, 1H), 6.39 (d, J=9.0 Hz, 1H), 6.31-6.27 (m, 1H),6.16-6.11 (m, 1H), 5.63 (s, 1H), 5.26 (s, 1H), 4.95-4.81 (m, 1H),4.20-411 (m, 1H), 3.95 (d, J=8.2 Hz, 1H), 3.84 (d, J=12.4 Hz, 1H), 3.64(d, J=12.1 Hz, 1H); ¹⁹F-NMR (376 MHz, DMSO-d6): δ −201.00.

Preparation of (10): To a solution of 9 (1.6 g, 6.9 mmol) in pyridine(20.0 mL) was added DMTrCl (3.5 g, 10.5 mmol) at 20° C. and stirred for1 h. TLC showed 9 was consumed completely. Water was added and extractedwith EA, the organic layer was washed with NaHCO₃ and brine. Then thesolution was concentrated under reduced pressure and the residue waspurified by Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=4/1 within 25 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1;Detector, UV 254 nm. This resulted in to give 10 (2.2 g, 4.2 mmol, 60.8%yield) as a white solid. ESI-LCMS: m/z 530.1 [M−H]⁻; ¹H-NMR (400 MHz,DMSO-d6): δ 7.93-7.91 (m, 1H), 7.47-7.23 (m, 10H), 6.91-6.89 (m, 4H),6.41 (d, J=8.8 Hz, 1H), 6.13 (d, J=18.8 Hz, 1H), 6.00-5.96 (m, 1H), 5.68(d, J=6.6 Hz, 1H), 5.01 (d, J=4.2 Hz, 0.5H), 4.88 (d, J=4.2 Hz, 0.5H),4.42-4.31 (m, 1H), 4.10-4.08 (m, 1H), 3.74 (s, 6H), 3.40-3.34 (m, 2H);¹⁹F-NMR (376 MHz, DMSO-d6): δ −199.49.

Preparation of Example 45 monomer: To a solution of 10 (2.2 g, 4.2 mmol)in DCM (20.0 mL) was added DCI (415 mg, 3.5 mmol) and CEP (1.5 g, 4.9mmol) under N₂ pro. The mixture was stirred at 20° C. for 0.5 h. TLCshowed 10 was consumed completely. The product was extracted with DCM,the organic layer was washed with H₂O and brine. Then the solution wasconcentrated under reduced pressure and the residue was purified by cc(PE/EA=5:1-1:1) and Flash-Prep-HPLC with the following conditions(IntelFlash-1): Column, C18 silica gel; mobile phase, CH₃CN/H₂O (0.5%NH₄HCO₃)=1/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O within 25 min,the eluted product was collected at CH₃CN/H₂O (0.5% NH₄HCO₃)=I/O;Detector, UV 254 nm. This resulted in to give Example 45 monomer (2.1 g,3.0 mmol, 73.1% yield) as a white solid. ESI-ESI-LCMS: m/z 732.2 [M+H]⁺;¹H-NMR (400 MHz, DMSO-d6): δ 7.98-7.92 (m, 1H), 7.42-7.24 (m, 10H),6.91-6.85 (m, 4H), 6.43-6.39 (m, 1H), 6.18-6.11 (m, 1H), 6.01-5.97 (m,1H), 5.22-5.19 (m, 0.5H), 5.09-5.06 (m, 0.5H), 4.73-4.52 (m, 1H),4.21-4.19 (m, 1H), 3.79-3.62 (m, 7H), 3.57-3.47 (m, 4H), 3.32-3.28 (m,1H), 2.75-2.58 (m, 1H), 1.13-0.92 (m, 12H); ¹⁹F-NMR (376 MHz, DMSO-d6):δ −196.82 , −196.84 , −197.86 , −197.88; ³¹P-NMR (162 MHz, DMSO-d6): δ149.88, 149.83, 149.39, 149.35.

Example 46. Synthesis of Monomer

Preparation of (2): To the solution of Bromobenzene (2.1 g, 13.6 mmol)in dry THF (15 mL) was added 1.6 M n-BuLi (7 mL, 11.8 mmol) drop wise at−78° C. The mixture was stirred at −78° C. for 0.5 h. Then the 1 (3.0 g,9.1 mmol, Wang, Guangyi et al, Journal of Medicinal Chemistry, 2016,59(10), 4611-4624) was dissolved in THF (15 mL) and added to the mixturedrop wise with keeping at −78° C. Then the reaction mixture was stirredat −78° C. for 1 hr. LC-MS showed 1 was consumed completely. Then thesolution was added to saturated aq. NH₄Cl and the resulting mixture wasextracted with EA. The combined organic layer was washed with water andbrine, dried over Na₂SO₄, and concentrated under reduced pressure togive a residue which was purified by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=4/1within 25 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=3/2; Detector, UV 254 nm. This resulted in to give 2 (3.0 g,7.3 mmol, 80.0%) as a white solid. ESI-LCMS: m/z 391 [M-OH]⁻.

Preparation of (3): To the solution of 2 (4.0 g, 9.8 mmol) in DCM (40mL) was added TES (1.9 g, 11.7 mmol) at −78° C., and the mixture wasadded BF₃.OEt₂ (2.1 g, 14.7 mmol) drop wise at −78° C. The mixture wasstirred at −40° C. for 1 hr. LC-MS showed 2 was consumed completely.Then the solution was added to saturated aq. NaHCO₃ and the resultingmixture was extracted with DCM. The combined organic layer was washedwith water and brine, dried over Na₂SO₄, and concentrated under reducedpressure to give a residue which was purified by Flash-Prep-HPLC withthe following conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=2/3 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=4/1 within 25 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=7/3; Detector, UV 254 nm. This resulted in togive 3 (3.1 g, 5.3 mmol, 54.0%) as a water clear oil. ESI-LCMS: m/z 410[M+H₂O]⁺; ¹H-NMR (400 MHz, CDCl₃: S 7.48-7.25 (m, 15H), 5.24-5.13 (m,1H), 4.93-4.74 (m, 1H), 4.74-4.46 (m, 4H), 4.37-4.25 (m, 1H), 4.19-4.05(m, 1H), 4.00-3.80 (m, 1H), 3.77-3.63 (m, 1H). ¹⁹F-NMR (376 MHz, CDCl₃):δ −196.84.

Preparation of (4): To the solution of 3 (2.1 g, 5.3 mmol) in dry DCM(20 mL) was added 1 M BCl₃ (25 mL, 25.5 mmol) drop wise at −78° C., andthe reaction mixture was stirred at −78° C. for 0.5 hr. LC-MS showed 3was consumed completely. After completion of reaction, the resultingmixture was poured into water (50 mL). The solution was extracted withDCM and the combined organic layer was concentrated under reducedpressure to give a crude. The crude in MeOH (4 mL) was added 1 M NaOH(15 mL), and the mixture was stirred at r.t for 5-10 min. The mixturewas extracted with EA. The combined organic layer was washed with brine,dried over Na₂SO₄, and concentrated under reduced pressure to give aresidue which was purified by silica gel column chromatography (eluent,DCM: MeOH=40:1˜15:1) to give 4 (1.0 g, 4.7 mmol, 88.6%) as a water clearoil. ESI-LCMS: m/z 211 [M−H]⁻; ¹H-NMR (400 MHz, DMSO-d6): δ 7.58-7.19(m, 5H), 5.41 (d, J=6.1 Hz, 1H), 5.09-5.95 (m, 1H), 5.95-4.84 (m, 1H),4.82-4.59 (m, 1H), 4.14-3.94 (m, 1H), 3.89-3.80 (m, 1H), 3.78-3.67 (m,1H), 3.65-3.53 (m, 1H). ¹⁹F-NMR (376 MHz, DMSO-d6): δ −196.46.

Preparation of (5): To a solution of 4 (1.0 g, 4.7 mmol) in Pyridine (10mL) was added DMTrCl (2.0 g, 5.7 mmol). The reaction mixture was stirredat r.t. for 2 hr. LCMS showed 4 was consumed and water (100 mL) wasadded. The product was extracted with EA (100 mL) and the organic layerwas washed with brine and dried over Na₂SO₄ and concentrated to give thecrude. The crude was further purified by Flash-Prep-HPLC with thefollowing conditions (IntelFlash-1): Column, C18 silica gel; mobilephase, CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O within 20 min, the eluted product was collected atCH₃CN/H₂O (0.5% NH₄HCO₃)=9/1; Detector, UV 254 nm. This resulted in togive 5 (2.1 g, 4.1 mmol, 87.0%) as a red oil. ESI-LCMS: m/z 513 [M−H]⁻;¹H-NMR (400 MHz, DMSO-d6): δ 7.56-7.16 (m, 14H), 6.94-9.80 (m, 4H), 5.45(d, J=6.3 Hz, 1H), 5.21-5.09 (m, 1H), 4.89-4.68 (m, 1H), 4.18-4.03 (m,2H), 3.74 (s, 6H), 3.33-3.29 (m, 1H), 3.26-3.17 (m, 1H). ¹⁹F-NMR (376MHz, DMSO-d6): δ −194.08.

Preparation of Example 46 monomer: To a suspension of 5 (2.1 g, 4.1mmol) in DCM (20 mL) was added DCI (410 mg, 3.4 mmol) and CEP[N(iPr)₂]₂(1.5 g, 4.9 mmol). The mixture was stirred at r.t. for 1 h. LC-MS showed5 was consumed completely. The solution was washed with water twice andwashed with brine and dried over Na₂SO₄. Then concentrated to give thecrude. The crude was purification by Flash-Prep-HPLC with the followingconditions (IntelFlash-1): Column, C18 silica gel; mobile phase,CH₃CN/H₂O (0.5% NH₄HCO₃)=1/1 increasing to CH₃CN/H₂O (0.5% NH₄HCO₃)=I/Owithin 20 min, the eluted product was collected at CH₃CN/H₂O (0.5%NH₄HCO₃)=I/O; Detector, UV 254 nm. This resulted in to give Example 46monomer (2.1 g, 2.9 mmol, 70.0%) as a white solid. ESI-LCMS: m/z 715[M+H]⁺; H-NMR (400 MHz, DMSO-d6): δ 7.59-7.16 (m, 14H), 6.94-9.80 (m,4H), 5.26-5.12 (m, 1H), 5.06-4.77 (m, 1H), 4.50-4.20 (m, 1H), 4.20-4.10(m, 1H), 3.83-3.63 (m, 7H), 3.59-3.37 (m, 4H), 3.25-3.13 (m, 1H),2.80-2.66 (m, 1H), 2.63-2.53 (m, 1H), 1.18-0.78 (m, 12H). ¹⁹F-NMR (376MHz, DMSO-d6): δ −194.40 , −194.42 , −194.50,-194.53. ³¹P-NMR (162 MHz,DMSO-d6): δ 149.38, 149.30, 149.02, 148.98.

Example 47. Deuterated Vinyl Phosphonate Improves Potency of siNA

This example investigates whether a deuterated vinyl phosphonateimproves potency of siNA in an AAV-HBV mouse. AAV-HBV mice weresubcutaneously injected with vehicle, ds-siNA-0165 (e.g., siNA without adeuterated vinyl phosphonate), or ds-siNA-0144 (e.g., siNA with adeuterated vinyl phosphonate). For siNA-treated AAV-HBV mice, AAV-HBVmice were subcutaneously injected with a single dose of 5 mg/kg of siNA.As shown in FIG. 11 , siNA molecules having 2′-fluoro nucleotides atpositions 3, 7-9, 12, and 17 from the 5′ end of the sense strand and2′-fluoro nucleotides at positions 2 and 14 from the 5′ end of theantisense strand resulted in at least a 0.5-log reduction in HBsAg, withthe greatest reduction in HBsAg found in mice treated with thedeuterated vinylphopshonate siNA (ds-siNA-0165). Thus, FIG. 11demonstrates that the presence of a deuterated vinyl phosphonateimproves potency of the siNA.

Example 48. Deuterated Vinyl Phosphonate Results in a Greater Reductionin Serum HBsAg

AAV-HBV mice were subcutaneously injected with vehicle, ds-siNA-0163(e.g., siNA without a vinyl phosphonate), ds-siNA-0122 (e.g., siNA witha vinyl phosphonate), or ds-siNA-0123 (e.g., siNA with a deuteratedvinyl phosphonate). For siNA-treated AAV-HBV mice, AAV-HBV mice weresubcutaneously injected with a single dose of 5 mg/kg of siNA. As shownin FIG. 12 , siNA molecules having 2′-fluoro nucleotides at positions 7,9-11 from the 5′ end of the sense strand and 2′-fluoro nucleotides atpositions 2 and 14 from the 5′ end of the antisense strand resulted inat least a 0.5-log reduction in HBsAg, with the greatest reduction inHBsAg found in mice treated with the deuterated vinylphopshonate siNA(ds-siNA-0165). Thus, FIG. 12 demonstrates that the presence of adeuterated vinyl phosphonate improves potency of the siNA, as comparedto the siNA without a vinyl phosphonate and the siNA with the vinylphosphonate.

Example 49: Synthesis of 5′ End Cap Monomer

Example 49 Monomer Synthesis Scheme

Preparation of(2): 1(15 g, 58.09 mmol) and tert-butylN-methylsulfonylcarbamate (17.01 g, 87.13 mmol) were dissolved in THF(250 mL), and PPh₃ (30.47 g, 116.18 mmol) was added followed by dropwiseaddition of DIAD (23.49 g, 116.18 mmol, 22.59 mL) at 0° C. The reactionmixture was stirred at 15° C. for 12 h. Upon completion as monitored byTLC(DCM/MeOH=10/1), the reaction mixture was evaporated to give aresidue. The residue was purified by flash silica gel chromatography(ISCO@; 120 g SepaFlash@ Silica Flash Column, Eluent of 0˜20%/MeOH/DCMgradient @ 60 mL/min) to give 2 (6.9 g, 24.28% yield) as a white solid.ESI-LCMS: m/z 457.9 [M+Na]⁺; ¹H NMR (400 MHz, CDCl₃) δ=8.64 (br s, 1H),7.64 (d, J=8.2 Hz, 1H), 5.88 (d, J=1.9 Hz, 1H), 5.80 (dd, J=2.2, 8.2 Hz,1H), 4.19-4.01 (m, 3H), 3.90 (dt, J=5.5, 8.2 Hz, 1H), 3.82-3.78 (m, 1H),3.64 (s, 3H), 3.32 (s, 3H), 2.75 (d, J=8.9 Hz, 1H), 1.56 (s, 9H).

Preparation of (3): 2 (6.9 g, 15.85 mmol) was dissolved in MeOH (40 mL),and a solution of HCl/MeOH (4 M, 7.92 mL) was added dropwise. Thereaction mixture was stirred at 15° C. for 12 h, and then evaporated togive a residue. The residue was purified by flash silica gelchromatography (ISCO®; 40 g SepaFlash@ Silica Flash Column, Eluent of0˜10% MeOH/DCM gradient @ 40 mL/min) to give 3 (2.7 g, 50.30% yield) asa white solid. ESI-LCMS: m/z 336.0 [M+H]⁺; ¹H NMR (400 MHz, CD3CN)δ=9.20 (br s, 1H), 7.52 (d, J=8.1 Hz, 1H), 5.75 (d, J=3.8 Hz, 1H), 5.64(dd, J=2.0, 8.1 Hz, 1H), 5.60-5.52 (m, 1H), 4.15-3.99 (m, 1H), 3.96-3.81(m, 2H), 3.46 (s, 3H), 3.44-3.35 (m, 1H), 3.34-3.26 (m, 1H), 2.92 (s,3H).

Preparation of (Example 49 monomer): To a solution of 3 (2.14 g, 6.38mmol) in DCM (20 mL) was added dropwise3-bis(diisopropylamino)phosphanyloxypropanenitrile (2.50 g, 8.30 mmol,2.63 mL) at 0° C., followed by 1H-imidazole-4, 5-dicarbonitrile (829 mg,7.02 mmol), and the mixture was purged under Ar for 3 times. Thereaction mixture was stirred at 15° C. for 2 h. Upon completion, themixture was quenched with 5% NaHCO₃(20 mL), extracted with DCM (20mL*2), washed with brine (15 mL), dried over Na₂SO₄, filtered, andevaporated to give a residue. The residue was purified by flash silicagel chromatography (ISCO®; 40 g SepaFlash@ Silica Flash Column, Eluentof 0˜10% (Phase B: i-PrOH/DCM=1/2)/Phase A: DCM with 5% TEA gradient @40 mL/min) to give Example 49 monomer (1.73 g, 48.59% yield) as a whitesolid. ESI-LCMS: m/z 536.3 [M+H]⁺; ¹H NMR (400 MHz, CD3CN) δ=7.58-7.48(m, 1H), 5.83-5.78 (m, 1H), 5.71-5.64 (m, 1H), 4.40-4.29 (m, 1H),4.19-4.07 (m, 1H), 3.98 (td, J=5.3, 13.3 Hz, 1H), 3.90-3.78 (m, 2H),3.73-3.59 (m, 3H), 3.41 (d, J=14.8 Hz, 4H), 2.92 (br d, J=7.0 Hz, 3H),2.73-2.63 (m, 2H), 1.23-1.11 (m, 12H); ³¹P NMR (162 MHz, CD3CN)δ=149.81, 150.37.

Example 50: Synthesis of 5′ End Cap Monomer

Example 50 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1(10 g, 27.16 mmol) in DMF (23 mL)were added imidazole (3.70 g, 54.33 mmol) and ThSCI (8.19 g, 54.33 mmol)at 25 C. The mixture was stirred at 25° C. for 2 hr. Upon completion,the reaction mixture was diluted with H₂O (20 mL) and extracted with EA(30 mL * 2). The combined organic layers were washed with brine (20 mL *2), dried over Na₂SO₄, filtered and concentrated under reduced pressureto give 2 (13 g 99.2% yield) as a white solid. ESI-LCMS: m/z 482.9[M+H]1.

Preparation of (3): To a solution of 2 (35.00 g, 72.56 mmol) in DMF (200mL) was added NaN₃ (14.15 g, 217.67 mmol). The mixture was stirred at60° C. for 17 h. Upon completion, the reaction mixture was diluted withH₂O (200 mL) and extracted with EA (200 mL*2). The combined organiclayers were washed with brine (100 mL * 2), dried over Na₂SO₄, filteredand concentrated under reduced pressure to give 3 (31.8 g, crude) as ayellow solid. ESI-LCMS: m/z 398.1 [M+H]+; ¹H NMR (400 MHz, DMSO-d6)δ=11.21 (d, J=1.3 Hz, 1H), 7.50 (d, J=8.1 Hz, 1H), 5.57 (d, J=4.5 Hz,1H), 5.46 (dd, J=2.1, 8.0 Hz, 1H), 4.06 (t, J=5.2 Hz, 1H), 3.81-3.64 (m,2H), 3.44-3.30 (m, 2H), 2.31-2.25 (m, 3H), 0.65 (s, 9H), −0.13 (s, 6H).

Preparation of (4): To a solution of 3 (7 g, 17.61 mmol) in THF (60 mL)was added Pd/C (2 g) at 25° C. The reaction mixture was stirred at 25°C. for 3 h under H2 atmosphere (15 PSI). The reaction mixture wasfiltered, and the filtrate was concentrated to give 4 (5.4 g, 75.11%yield) as a gray solid. ESI-LCMS: m/z 372.1 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d6) δ=7.93 (d, J=8.0 Hz, 1H), 5.81 (d, J=5.5 Hz, 1H), 5.65 (d,J=8.3 Hz, 1H), 4.28 (t, J=4.6 Hz, 1H), 3.88 (t, J=5.3 Hz, 1H), 3.74 (q,J=4.6 Hz,1 H), 3.31 (s, 3H), 2.83-2.66 (m, 2H), 0.88 (s, 9H), 0.09 (s,6H).

Preparation of (5): To a solution of 4 (3 g, 8.08 mmol) in DCM (30 mL)was added TEA (2.45 g, 24.23 mmol, 3.37 mL) followed by dropwiseaddition of 3-chloropropane-1-sulfonyl chloride (1.50 g, 8.48 mmol, 1.03mL) at 25° C. The reaction mixture was stirred at 25° C. for 18 h underN₂ atmosphere. Upon completion, the reaction mixture was diluted withH₂O (50 mL) and extracted with DCM (50 mL * 2). The combined organiclayers were washed with brine (50 mL*2), dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashsilica gel chromatography (ISCO@; 24 g SepaFlash@ Silica Flash Column,Eluent of 0˜30% MeOH/DCM @ 50 mL/min) to give 5 (3.6 g, 84.44% yield) asa white solid. ESI-LCMS: m/z 512.1 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6)δ=11.42 (s, 1H), 7.75 (d, J=8.1 Hz, 1H), 7.49 (t, J=6.2 Hz, 1H), 5.83(d, J=5.8 Hz, 1H), 5.70-5.61 (m, 1H), 4.33-4.23 (m, 1H), 3.95 (t, J=5.5Hz, 1H), 3.90-3.78 (m, 1H), 3.73 (t, J=6.5 Hz, 2H), 3.30 (s, 3H),3.26-3.12 (m, 4H), 2.14-2.02 (m, 2H), 0.88 (s, 9H), 0.11 (d, J=3.3 Hz,6H).

Preparation of (6): To a solution of 5 (5 g, 9.76 mmol) in DMF (45 mL)was added DBU (7.43 g, 48.82 mmol, 7.36 mL). The mixture was stirred at25° C. for 16 h. The reaction mixture was concentrated to give aresidue. diluted with H₂O (50 mL) and extracted with EA (50 mL * 2). Thecombined organic layers were washed with brine (50 mL * 2), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas purified by flash silica gel chromatography (ISCO®; 24 g SepaFlash@Silica Flash Column, Eluent of 0˜80% EA/PE @ 40 mL/min) to give 6 (4.4g, 89.06% yield) as a white solid. ESI-LCMS: m/z 476.1 [M+H]⁺; H NMR(400 MHz, DMSO-d6) δ=11.43 (d, 1=1.7 Hz, 1H), 7.72 (d, J=8.1 Hz, 1H),5.82 (d, J=4.8 Hz, 1H), 5.67 (dd, J=2.1, 8.1 Hz, 1H), 4.22 (t, J=5.1 Hz,1H), 3.99-3.87 (m, 2H), 3.33-3.27 (m, 6H), 3.09 (dd, J=6.6, 14.7 Hz,1H), 2.26-2.16 (m, 2H), 0.88 (s, 9H), 0.10 (d, J=3.8 Hz, 6H).

Preparation of (7): To a solution of 6 (200 mg, 420.49 umol) in MeOH (2mL) was added NH₄F (311.48 mg, 8.41 mmol, 20 eq), and the mixture wasstirred at 80° C. for 2 h. The mixture was filtered and concentrated togive a residue, which was purified by flash silica gel chromatography(ISCO®; 4 g SepaFlash@ Silica Flash Column, Eluent of 0˜50% MeOH/DCM @20 mL/min) to give 7 (120 mg, 76.60% yield) as a white solid. ESI-LCMS:m/z 362.1 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ=11.37 (br s, 1H), 7.68 (d,J=8.1 Hz, 1H), 5.81 (d, J=4.6 Hz, 1H), 5.65 (d, J=8.0 Hz, 1H), 4.02 (q,J=5.6 Hz,1 H), 3.95-3.83 (m, 2H), 3.34 (s, 9H), 3.09 (dd, J=6.9, 14.6Hz, 1H), 2.26-2.14 (m, 2H).

Preparation of (Example 50 monomer): To a solution of 7 (1.5 g, 4.15mmol) in CH₃CN (12 mL) were added3-bis(diisopropylamino)phosphanyloxypropanenitrile (1.63 g, 5.40 mmol,1.71 mL) and 1H-imidazole-4,5-dicarbonitrile (539.22 mg, 4.57 mmol) inone portion at 0° C. The reaction mixture was gradually warmed to 25° C.The reaction mixture was stirred at 25° C. for 2 h under N₂ atmosphere.Upon completion, the reaction mixture was diluted with NaHCO₃(20 mL) andextracted with DCM (20 mL * 2). The combined organic layers were washedwith brine (20 mL * 2), dried over Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue, which was purified by flashsilica gel chromatography (ISCO®; 12 g SepaFlash@ Silica Flash Column,Eluent of 0-85% EA/PE with 0.5% TEA @ 30 mL/min to give Example 50monomer (800 mg, 33.6% yield,) as a white solid. ESI-LCMS: m/z 562.3[M+H]⁺; ¹H NMR (400 MHz, CD3CN) δ=9.28 (br s, 1H), 7.55 (br dd, J=8.3,12.8 Hz,1 H), 5.86 (br d, J=3.9 Hz, 1H), 5.65 (br d, J=8.0 Hz, 1H),4.33-4.06 (m, 2H), 4.00-3.89 (m, 1H), 4.08-3.86 (m, 1H), 3.89-3.72 (m,4H), 3.43 (br d, J=15.1 Hz, 6H), 3.23-3.05 (m, 3H), 2.69 (br s, 2H),2.36-2.24 (m, 2H), 1.26-1.10 (m, 12H); ³¹P NMR (162 MHz, CD3CN)δ=149.94, 149.88.

Example 51: Synthesis of 5′ End Cap Monomer

Example 51 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1(30 g, 101.07 mmol, 87% purity) inCH₃CN (1.2 L) and Py (60 mL) were added I₂ (33.35 g, 131.40 mmol, 26.47mL) and PPh₃ (37.11 g, 141.50 mmol) in one portion at 10° C. Thereaction was stirred at 25° C. for another 48 h. The mixture was dilutedwith aq.Na₂S203 (300 mL) and aq.NaHCO₃(300 mL), concentrated to removeCH₃CN, and then extracted with EtOAc (300 mL * 3). The combined organiclayers were washed with brine (300 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by flash silica gel chromatography (ISCO®; 330 g SepaFlash@Silica Flash Column, Eluent of 0.60%/Methanol/Dichloromethane gradient @100 mL/min) to give 2 (28.2 g, 72.00% yield, 95% purity) as a brownsolid. ESI-LCMS: m/z 369.1 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ=11.43 (s,1H), 7.68 (d, J=8.1 Hz, 1H), 5.86 (d, J=5.5 Hz, 1H), 5.69 (d, J=8.1 Hz,1H), 5.46 (d, J=6.0 Hz, 1H), 4.08-3.96 (m, 2H), 3.90-3.81 (m, 1H),3.60-3.51 (m, 1H), 3.40 (dd, J=6.9, 10.6 Hz, 1H), 3.34 (s, 3H).

Preparation of (3): To a solution of 2 in DMF (90 mL) were addedimidazole (4.25 g, 62.48 mmol) and TBSCI (6.96 g, 46.18 mmol) in oneportion at 15° C. The mixture was stirred at 15° C. for 6 h. Thereaction mixture was quenched by addition of H₂O (300 mL) and extractedwith EtOAc (300 mL * 2). The combined organic layers were washed withbrine (300 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give 3 (13.10 g, crude) as a white solid. ESI-LCMS:m/z 483.0 [M+H]⁺.

Preparation of (4): To a solution of 3 (10 g, 20.73 mmol) in MeOH (20mL), H₂O (80 mL), and dioxane (20 mL) was added Na₂SO₃ (15.68 g, 124.38mmol), and the mixture was stirred at 80° C. for 24 h. The reactionmixture was concentrated under reduced pressure to remove MeOH. Theaqueous layer was extracted with EtOAc (80 mL * 2) and concentratedunder reduced pressure to give a residue. The residue was trituratedwith MeOH (100*3 mL) to give 4 (9.5 g, 94.48% yield, 90% purity) as awhite solid. ESI-LCMS: m/z 437.0 [M+H]⁺.

Preparation of (5): To a solution of 4 (11 g, 21.42 mmol, 85% purity) inDCM (120 mL) was added DMF (469.65 mg, 6.43 mmol, 494.37 uL) at 0° C.,followed by dropwise addition of oxalyl dichloride (13.59 g, 107.10mmol, 9.37 mL). The mixture was stirred at 20° C. for 2 h. The reactionmixture was quenched by addition of water (60 mL) and the organic layer5 (0.1125 M, 240 mL DCM) was used directly for next step. (This reactionwas set up for two batches and combined) ESI-LCMS: m/z 455.0 [M+H]⁺.

Preparation of (6): 5 (186.4 mL, 0.1125 M in DCM) was diluted with DCM(60 mL) and treated with methylamine (3.26 g, 41.93 mmol, 40% purity).The mixture was stirred at 20° C. for 2 h. The reaction mixture wasconcentrated under reduced pressure to give a residue. The residue waspurified by flash silica gel chromatography (ISCO®; 40 g SepaFlash@Silica Flash Column, Eluent of 0˜10%, MeOH/DCM gradient @ 40 mL/min) togive AGS-9-3-008 (1.82 g, 18.53% yield, 96% purity) as a yellow solid.ESI-LCMS: m/z 472.0 [M+Na]⁺; ¹H NMR (400 MHz, CDCl₃) δ=9.08 (s, 1H),7.31 (d, J=8.1 Hz, 1H), 5.78 (d, J=8.1 Hz, 1H), 5.57 (d, J=3.8 Hz, 1H),4.61-4.48 (m, 1H), 4.41-4.27 (m, 2H), 4.13-4.03 (m, 1H), 3.46 (s, 3H),3.43-3.33 (m, 2H), 2.78 (d, J=5.2 Hz, 3H), 0.92 (s, 9H), 0.13 (s, 6H).

Preparation of (7): To a solution of 6 (2.3 g, 5.12 mmol) in MeOH (12mL) was added HCl/MeOH (4 M, 6.39 mL). The mixture was stirred at 20° C.for 2 h. The reaction mixture was concentrated under reduced pressure togive a residue. The residue was purified by flash silica gelchromatography (ISCO®; 24 g SepaFlash@ Silica Flash Column, Eluent of0-15%, MeOH/DCM gradient @ 30 mL/min) to give 7 (1.4 g, 79.98% yield) asa pink solid. ESI-LCMS: m/z 336.1 [M+H]⁺; ¹H NMR (400 MHz, CDCl₃) δ=9.12(s, 1H), 7.39 (d, J=8.0 Hz, 1H), 5.79 (d, J=3.3 Hz, 1H), 5.66 (dd,J=2.1, 8.2 Hz, 1H), 5.13 (s, 1H), 4.13 (t, J=4.0, 7.4 Hz, 1H), 4.07-4.02(m, 1H), 3.87 (dd, J=3.3, 5.5 Hz, 1H), 3.47 (s, 3H), 3.43-3.37 (m, 2H),2.65 (d, J=4.5 Hz, 3H).

Preparation of (Example 51 monomer): To a mixture of 7 (1.7 g, 5.07mmol) and 4A MS (1.4 g) in MeCN (18 mL) was added3-bis(diisopropylamino)phosphanyloxypropanenitrile (1.99 g, 6.59 mmol,2.09 mL) at 0° C., followed by addition of1H-imidazole-4,5-dicarbonitrile (658.57 mg, 5.58 mmol) in one portion at0° C. The mixture was stirred at 20° C. for 2 h. Upon completion, thereaction mixture was quenched by addition of sat. NaHCO₃ solution (20mL) and diluted with DCM (40 mL). The organic layer was washed with sat.NaHCO₃(20 mL * 2), dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. The residue was purified by a flashsilica gel column (0% to 5% i-PrOH in DCM with 5% TEA) to give Example51 monomer (1.30 g, 46.68% yield) as a white solid. ESI-LCMS: m/z 536.2[M+H]⁺; ¹H NMR (400 MHz, CD3CN) δ=9.00 (s, 1H), 7.40 (d, J=8.0 Hz, 1H),5.85-5.76 (m, 1H), 5.64 (d, J=8.0 Hz, 1H), 5.08 (d, J=5.0 Hz, 1H),4.42-4.21 (m, 2H), 4.00 (td, J=4.6, 9.3 Hz, 1H), 3.89-3.61 (m, 4H),3.47-3.40 (m, 4H), 3.37-3.22 (m, 1H), 2.71-2.60 (m, 5H), 1.21-1.16 (m,11H), 1.21-1.16 (m, 1H); ³¹P NMR (162 MHz, CD3CN) δ=150.07, 149.97

Example 52: Synthesis of 5′ End Cap Monomer

Example 52 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1(13.10 g, 27.16 mmol) in THF (100mL) was added DBU (20.67 g, 135.78 mmol, 20.47 mL). The mixture wasstirred at 60° C. for 6 h. Upon completion, the reaction mixture wasquenched by addition of sat. NH₄Cl solution (600 mL) and extracted withEA (600 mL * 2). The combined organic layers were washed with brine (100ml), dried over Na₂SO₄, filtered and concentrated under reduced pressureto give a residue. The residue was purified by flash silica gelchromatography (ISCO@; 120 g SepaFlash@ Silica Flash Column, Eluent of0˜50% (Phase B: ethyl acetate: dichloromethane=1:1)/Phase A: petroleumethergradient@ 45 mL/min) to give 2 (5.9 g, 60.1% yield,) as a whitesolid. ESI-LCMS: m/z 355.1 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6)δ=11.61-11.30 (m, 1H), 7.76-7.51 (m, 1H), 6.04 (d, J=5.4 Hz, 1H), 5.75(s, 1H), 5.73-5.67 (m, 1H), 4.78 (d, J=4.9 Hz, 1H), 4.41 (d, J=1.1 Hz,1H), 4.30 (t, J=4.8 Hz, 1H), 4.22 (d, J=1.4 Hz, 1H), 4.13 (t, J=5.1 Hz,1H), 4.06-3.97 (m, 1H), 3.94-3.89 (m, 1H), 3.82-3.75 (m, 1H), 3.33 (s,3H), 3.30 (s, 2H), 1.17 (t, J=7.2 Hz, 1H), 0.89 (s, 9H), 0.16-0.09 (m,6H).

Preparation of (3): To a solution of 2 (4 g, 11.28 mmol) in DCM (40 mL)was added Ru(II)-Pheox (214.12 mg, 338.53 umol) in one portion followedby addition of diazo(dimethoxyphosphoryl)methane (2.54 g, 16.93 mmol)dropwise at 0° C. under N₂. The reaction was stirred at 20° C. for 16 h.Upon completion, the reaction mixture was filtered and concentratedunder reduced pressure to give a residue. The residue was purified byflash silica gel chromatography (ISCO®; 80 g SepaFlash@ Silica FlashColumn, Eluent of 0-4% MeOH/DCM@ 60 mL/min) to give 3 (5 g, 86.47%yield) as a red liquid. ESI-LCMS: m/z 477.1 [M+H]⁺; ¹H NMR (400 MHz,DMSO-d6) δ=11.46 (s, 1H), 7.49 (d, .,8.0 Hz, 1H), 6.01-5.87 (m, 1H),5.75 (dd, J=2.0, 8.0 Hz, 1H), 4.58 (d, .,3.8 Hz, 1H), 4.23 (dd, J=3.8,7.8 Hz, 1H), 3.80-3.68 (m, 6H), 3.30 (s, 3H), 1.65-1.46 (m, 2H),1.28-1.16 (m, 1H), 0.91 (s, 9H), 0.10 (d, J=4.3 Hz, 6H); ³¹P NMR (162MHz, DMSO-d6) δ=27.5

Preparation of (4): To a mixture of 3 (2.8 g, 5.88 mmol) and NaI (1.76g, 11.75 mmol) in CH₃CN (30 mL) was added chloromethyl2,2-dimethylpropanoate (2.21 g, 14.69 mmol, 2.13 mL) at 25° C. Themixture was stirred at 80° C. for 40 h under Ar. The reaction mixturewas filtered and concentrated under reduced pressure to give a residue.The residue was purified by flash silica gel chromatography (ISCO®; 40 gSepaFlash@ Silica Flash Column, Eluent of 0˜50% Ethylacetate/Petroleumether gradient @ 40 mL/min) to give 4 (2.1 g, 51.23% yield, 97% purity)as a yellow solid. ESI-LCMS: 677.3 [M+H]⁺.

Preparation of (5): A mixture of 4 (2.09 g, 3.09 mmol) in H₂O (1.5 mL)and HCOOH (741.81 mg, 15.44 mmol, 6 mL) was stirred at 15° C. for 40 h.Upon completion, the reaction mixture was quenched by saturatedaq.NaHCO₃(300 mL) and extracted with EA (300 mL * 2). The combinedorganic layers were washed with brine (300 mL), dried over Na₂SO₄,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by flash silica gel chromatography (ISCO®; 20 gSepaFlash® Silica Flash Column, Eluent of 0-5% Methanol/Dichloromethane@45 mL/min) to give 5 (1.51 g, 85.19% yield) as a yellow solid. ESI-LCMS:585.1 [M+Na]⁺; ¹H NMR (400 MHz, DMSO-d6) δ=11.45 (d, J=1.8 Hz, 1H), 7.44(d, J=8.2 Hz, 1H), 6.04 (d, J=7.5 Hz, 1H), 5.78-5.51 (m, 6H), 4.39 (t,J=4.4 Hz, 1H), 4.15 (dd, J=4.3, 7.4 Hz, 1H), 4.03 (q, J=7.1 Hz, 1H),1.99(s, 1H), 1.66 (dd, J=8.6, 10.8 Hz, 1H), 1.55-1.29 (m, 2H), 1.18 (d,J=2.0 Hz, 18H).

Preparation of (Example 52 monomer): To a solution of 5 (2.5 g, 4.44mmol) in MeCN (30 mL) was added3-bis(diisopropylamino)phosphanyloxypropanenitrile (1.74 g, 5.78 mmol,1.84 mL) at 0° C., followed by 1H-imidazole-4,5-dicarbonitrile (577.36mg, 4.89 mmol) in one portion under Ar. The mixture was gradually warmedto 20° C. and stirred at 20° C. for 1 h. The reaction mixture wasquenched by addition of sat. NaHCO₃ solution (50 mL) and diluted withDCM (250 mL). The organic layer was washed with sat. NaHCO₃ solution (50mL * 2), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by a flash silicagel column (0% to 50% EA/PE with 0.5% TEA) to give Example 52 monomer(1.85 g, 54.1% yield) as a white solid. ESI-LCMS: 785.2 [M+Na]⁺;¹H NMR(400 MHz, CD3CN) δ=9.18 (s, 1H), 7.31 (d, J=8.3 Hz, 1H), 6.06 (d, J=7.8Hz, 1H), 5.72-5.60 (m, 5H), 4.85-4.76 (m, 1H), 4.27 (m, 1H), 3.93-3.64(m, 4H), 3.41 (d, J=16.6 Hz, 3H), 2.80-2.62 (m, 2H), 1.76-1.49 (m, 3H),1.23-1.19 (m, 30H); ³¹P NMR (162 MHz, CD3CN) δ=150.66 (s), 150.30,24.77, 24.66.

Example 53: Synthesis of 5′ End Cap Monomer

Example 53 Monomer Synthesis Scheme

Preparation of (2): To a solution of 1 (15 g, 137.43 mmol) in DCM (75mL) were added Boc₂O (31.49 g, 144.30 mmol, 33.15 mL) and DMAP (839.47mg, 6.87 mmol, 0.05 eq) at 0° C. The mixture was stirred at 20° C. for16 hr, and concentrated under reduced pressure to give 2 (29.9 g, crude)as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ=3.23 (s, 3H), 3.16 (s, 3H),1.51 (s, 9H).

Preparation of (3): To a solution of 2 (24.9 g, 118.99 mmol) in THF (250mL) was added n-BuLi (2.5 M, 47.60 mL) dropwise at −78° C. under Ar andstirred at −78° C. for 1 hr. P-3 (17.19 g, 118.99 mmol, 12.83 mL) wasadded at 0° C. and stirred for 1 hr. The reaction mixture was quenchedby saturated aq. NH₄Cl (100 mL), and then extracted with EA (100 mL*2).The combined organic layers were washed with brine (100 mL*2), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by flash silica gel chromatography(ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0-50%Ethylacetate/Petroleum ethergradient @ 65 mL/min) to give 3 (7.1 g,18.62% yield) as a yellow oil. ESI-LCMS: 339.9 [M+Na]⁺, NMR (400 MHz,CDCl₃) δ=4.12 (s, 1H), 4.08 (s, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.22(s, 3H), 1.51 (s, 9H).

Preparation of (5): To a mixture of 4 (15 g, 40.27 mmol) and PPTS (10.12g, 40.27 mmol) in DMSO (75 mL) was added EDCI (23.16 g, 120.81 mmol) at20° C. The mixture was stirred at 20° C. for 4 hr. The reaction mixturewas diluted with water (150 mL) and extracted with EA (150 mL*2). Thecombined organic layers were washed with brine (150 mL*2), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give 5 (12g, crude) as a white solid. ESI-LCMS: 371.2[M+H]⁺; ¹H NMR (400 MHz,CDCl₃) δ=9.77 (s, 1H), 7.62 (d, J=8.1 Hz, 1H), 5.83-5.76 (m, 2H), 4.53(d, J=4.3 Hz, 1H), 4.43 (br t, J=4.4 Hz, 1H), 3.95 (br t, J=4.7 Hz, 1H),3.47-3.35 (m, 5H), 0.92 (s, 9H), 0.13 (d, J=5.8 Hz, 6H).

Preparation of (6): To a solution of P4 (8.02 g, 25.27 mmol) in THF (40mL) was added n-BuLi (2.5 M, 8.42 mL) dropwise under Ar at −78° C., andthe mixture was stirred at −78° C. for 0.5 hr. A solution of 4 (7.8 g,21.05 mmol) in THF (40 mL) was added dropwise. The mixture was allowedto warm to 0° C. and stirred for another 2 hr. The reaction mixture wasquenched by saturated aq. NH₄Cl solution (80 mL) and extracted with EA(80 mL). The combined organic layers were washed with brine (80 mL * 2),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by flash silica gelchromatography (ISCO®; 80 g SepaFlash@ Silica Flash Column, Eluent of0-38% ethylacetate/petroleum ether gradient @ 60 mL/min) to give 7 (7.7g, 13.43 mmol, 63.8% yield) as a white solid. ESI-LCMS: 506.2 [M-tBu]⁺;¹H NMR (400 MHz, CDCl₃) δ=8.97 (s, 1H), 7.25 (d, J=8.3 Hz, 1H),6.95-6.88 (m, 1H), 6.87-6.81 (m, 1H), 5.83-5.77 (m, 2H), 4.58 (dd,J=4.4, 6.7 Hz, 1H), 4.05 (dd, J=5.0, 7.5 Hz, 1H), 3.82-3.77 (m, 1H),3.53 (s, 3H), 3.20 (s, 3H), 1.50 (s, 9H), 0.91 (s, 9H), 0.11 (d, J=2.5Hz, 6H).

Preparation of (7): To a solution of 6 (7.7 g, 13.71 mmol) in MeOH (10mL) was added HCl/MeOH (4 M, 51.40 mL) at 20° C. The mixture was stirredat 20° C. for 16 hr. Upon completion, the reaction mixture wasconcentrated under reduced pressure to remove MeOH. The residue waspurified by flash silica gel chromatography (ISCO®; 80 g SepaFlash@Silica Flash Column, Eluent of 0-4% MeOH/DCM @ 60 mL/min) to give 7 (4.1g, 86.11% yield) as a white solid. ESI-LCMS: 369.9 [M+Na]⁺; ¹H NMR (400MHz, DMSO-d6) δ=11.44 (s, 1H), 7.66 (d, J=8.3 Hz, 1H), 7.11 (q, J=4.9Hz, 1H), 6.69 (dd, J=6.0, 15.1 Hz, 1H), 6.56-6.47 (m, 1H), 5.82 (d,J=4.0 Hz, 1H), 5.67 (dd, J=2.0, 8.0 Hz, 1H), 5.56 (br s, 1H), 4.42 (t,J=6.1 Hz, 1H), 4.13 (t, J=5.8 Hz, 1H), 3.97 (t, J=4.8 Hz, 1H), 3.39 (s,3H), 2.48 (d, J=5.3 Hz, 3H)

Preparation of (8): To a solution of 7 (2.5 g, 7.20 mmol) in THF (25 mL)was added Pd/C (2.5 g, 10% purity) under H₂ atmosphere, and thesuspension was degassed and purged with H₂ for 3 times. The mixture wasstirred under H₂ (15 Psi) at 20° C. for 1 hr. Upon completion, thereaction mixture was filtered and concentrated under reduced pressure togive a residue. The residue was purified by flash silica gelchromatography (ISCO®; 25 g SepaFlash@ Silica Flash Column, Eluent of0-5% Ethylacetate/Petroleum ethergradient @ 50 mL/min) to give 8 (2.2 g,87.49% yield,) as a white solid. ESI-LCMS: 372.1 [M+Na]⁺; ¹H NMR (400MHz, DMSO-d6) δ=11.40 (s, 1H), 7.62 (d, J=8.0 Hz, 1H), 6.93 (q, J=4.9Hz, 1H), 5.76 (d, J=4.5 Hz, 1H), 5.66 (d, J=8.0 Hz, 1H), 5.26 (d, J=6.3Hz, 1H), 3.97 (q, J=5.9 Hz, 1H), 3.91-3.79 (m, 2H), 3.36 (s, 3H),3.14-3.00 (m, 2H), 2.56 (d, J=5.0 Hz, 3H), 2.07-1.87 (m, 2H).

Preparation of (Example 53 monomer): To a solution of 8 (2.2 g, 6.30mmol, 1 eq) in CH₃CN (25 mL) was added P-1 (2.47 g, 8.19 mmol, 2.60 mL,1.3 eq) at 0° C., and then 1H-imidazole-4,5-dicarbonitrile (818.07 mg,6.93 mmol, 1.1 eq) was added in one portion at 0° C. under Ar. Themixture was stirred at 20° C. for 2 hr. Upon completion, the reactionmixture was quenched by saturated aq. NaHCO₃(25 mL), and extracted withDCM (25 mL * 2). The combined organic layers were washed with brine (25mL * 2), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by flash silica gelchromatography (ISCO®; 40 g SepaFlash@ Silica Flash Column, Eluent of40-85% ethylacetate/petroleum ether gradient @ 40 mL/min) to giveExample 53 monomer (2.15 g, 61.32% yield) as a white solid. ESI-LCMS:572.2 [M+Na]⁺;¹H NMR (400 MHz, CD3CN) δ=9.32 (br s, 1H), 7.39 (d, J=8.1Hz, 1H), 5.82-5.75 (m, 1H), 5.66 (dd, J=0.7, 8.1 Hz, 1H), 5.14 (qd,J=4.9, 9.4 Hz, 1H), 4.24-4.02 (m, 2H), 3.99-3.93 (m, 1H), 3.90-3.60 (m,4H), 3.43 (d, J=17.5 Hz, 3H), 3.18-3.08 (m, 2H), 2.74-2.61 (m, 5H),2.19-2.11 (m, 1H), 2.09-1.98 (m, 1H), 1.19 (ddd, J=2.4, 4.0, 6.6 Hz,12H).³¹P NMR (162 MHz, CD3CN) δ=149.77 (s), 149.63 (br s).

Example 54. Long-Term Efficacy of siNA in an AAV-HBV Mouse Model

AAV/HBV is a recombinant AAV carrying replicable HBV genome. Takingadvantage of the highly hepatotropic feature of genotype 8 AAV, the HBVgenome can be efficiently delivered to the mouse liver cells. Infectionof immune competent mouse with AAV/HBV can result in long term HBVviremia, which mimics chronic HBV infection in patients. The AAV/HBVmodel can be used to evaluate the in vivo activity of various types ofanti-HBV agents. Mice were infected with AAV-HBV on day −28 of thestudy. AAV-HBV mice were subcutaneously injected with a single dose of 5mL/kg of vehicle or 5 mg/kg of ds-siNA-0147 on day 0. Serial bloodcollections were usually taken every 5 days on day 0, 5, 10, and 15,etc. until the termination of the study. Serum HBV S antigen (HBsAg) wasassayed through ELISA. FIG. 13 shows a graph of the change in serumHBsAg from AAV-HBV mice treated with vehicle (G 15) or ds-siNA-0147 (G19). As shown in FIG. 13 , ds-siNA-0147 was effective in reducing serumHBsAg levels and the reduction in serum HBsAg levels was observed forthe duration of the study (i.e., 100 days). Thus, FIG. 13 demonstratesthat ds-siNA-0147 is effective and durable after a single dose of 5mg/kg.

ds-siNA SEQ ID Strand Sequence ID NO: ds-siNA- Sense5′-mGpsmUpsmGmGfUmGfGfAfCm 438 0147 UmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc4-3′ Antisense 3′-mApsmGpsmCmAmCfCmAfCmCm 501UmGmAmAmGmAfGmAmGmUpsfUpsm A-5′

Example 55. Deuterated Vinyl Phosphonate Improves Potency of siNA

This example investigates whether a deuterated vinyl phosphonateimproves potency of siNA in an AAV-HBV mouse. AAV-HBV mice weresubcutaneously injected with vehicle, ds-siNA-0109 (e.g., siNA without adeuterated vinyl phosphonate), or ds-siNA-0172 (e.g., siNA with adeuterated vinyl phosphonate). AAV-HBV mice were subcutaneously injectedwith a single dose of 5 mL/kg of vehicle or 5 mg/kg of ds-siNA-0149 ords-siNA-0172 at day 0. Serial blood collections were usually taken every5 days on day 0, 5, 10, and 15, etc. until the termination of the study.Serum HBV S antigen (HBsAg) was assayed through ELISA.

As shown in FIG. 14 , siNA molecules having 2′-fluoro nucleotides atpositions 5 and 7-9 from the 5′ end of the sense strand and 2′-fluoronucleotides at positions 2, 5, 8, 14, and 17 from the 5′ end of theantisense strand resulted in greater than a 0.5-log reduction in HBsAg,with the greatest reduction in HBsAg found in mice treated with thedeuterated vinylphopshonate siNA (ds-siNA-0172). In addition, theduration of the reduction in serum HBsAg levels was significantly longerfor the deuterated vinylphosphonate siNA (ds-siNA-0172). Thus, FIG. 14demonstrates that the presence of a deuterated vinyl phosphonateimproves potency and durability of the siNA.

ds-siNA SEQ ID ID Strand Sequence NO: ds-siNA- Sense5′-mCpsmCpsmGmUfGmUfGfCfAmCmUmUmCmGmC 424 0109 mUmUmCmAp-ps2-GalNAc4Antisense 3′-mCpsmUpsmGmGfCmAmCfAmCmGmUmGmAfAmG 485 mCfGmAmApsfGpsmU-5′ds-siNA- Sense 5′-mCpsmCpsmGmUfGmUfGfCfAmCmUmUmCmGmC 424 0172mUmUmCmA-p-ps2-GalNAc4-3′ Antisense3′-mCpsmUpsmGmGfCmAmCfAmCmGmUmGmAfAmG 536 mCfGmAmApsfGpsd2vd3U-5′ d2vd3U=

Example 56. Comparison of siNAs

AAV/HBV is a recombinant AAV carrying replicable HBV genome. Takingadvantage of the highly hepatotropic feature of genotype 8 AAV, the HBVgenome can be efficiently delivered to the mouse liver cells. Infectionof immune competent mouse with AAV/HBV can result in long term HBVviremia, which mimics chronic HBV infection in patients. The AAV/HBVmodel can be used to evaluate the in vivo activity of various types ofanti-HBV agents. Mice were infected with AAV-HBV on day −28 of thestudy. AAV-HBV mice were subcutaneously injected with a single dose of 5mL/kg of vehicle or 5 mg/kg of ds-siNA-0109, ds-siNA-0119, ords-siNA-0153 on day 0. Serial blood collections were usually taken every5 days on day 0, 5, 10, and 15, etc. until the termination of the study.Serum HBV S antigen (HBsAg) was assayed through ELISA. FIG. 15 shows agraph of the change in serum HBsAg from AAV-HBV mice treated withvehicle (G 01, circle), ds-siNA-0109 (G 07, square), ds-siNA-0119 (G11,triangle), or ds-siNA-0153 (G13, diamond). As shown in FIG. 14 , allthree ds-siNAs were effective in reducing serum HBsAg levels and thereduction in serum HBsAg levels was observed for the duration of thestudy (i.e., 100 days), with the best potency and durability observedfor ds-siNA-0153. Thus, FIG. 15 demonstrates that ds-siNA-0109,ds-siNA-0119, and ds-siNA-0153 were effective and durable after a singledose of 5 mg/kg.

ds-siNA SEQ ID Strand Sequence ID NO: ds-siNA- Sense5′-mCpsmCpsmGmUfGmUfGfCfAm 424 0109 CmUmUmCmGmCmUmUmCmAp-ps2- GalNAc4Antisense 3′-mCpsmUpsmGmGfCmAmCfAmCm 485 GmUmGmAfAmGmCfGmAmApsfGpsm U-5′ds-siNA- Sense 5′-mGpsmCpsmUmGfCmUmAmUfGf 430 0119CfCmUmCfAmUmCmUmUfCmUmU- p-ps2-GalNAc4 Antisense3′-mGpsmApsmCmGmAmCmGmAmUf 595 AmCmGmGmAmGmUmAmGmAmAmGpsf ApsmA-5′ds-siNA- Sense 5′-mUpsmGpsfUmGmCmAfCfUfUm 441 0153CmGfCmUmUmCmAfCmCmU-p-ps2- GalNAc4-3′ Antisense3′-mGpsmCpsmAfCmAmCmGfUmGm 526 AmAfGmCmGmAfAmGmUmGpsfGpsm A-5

Example 57. Efficacy of a Combination Therapy in AAV-HBV Mouse Model

This example investigates the efficacy of a combination therapycomprising an antisense oligonucleotide (ASO 1, 5′GalNAc4-ps-GalNAc4-ps-GalNAc4-po-mA-po-lnGpslnApslnTpslnApslnApsApsAps(50H)CpsGps(5m)Cps(5 m)CpsGps(5 m)CpslnApslnG pslnApscp(5 m)C-3′(SEQ ID NO: 534)) anda ds-siNA-0147 for treating HBV in an AAV-HBV mouse model.

AAV-HBV mice were subcutaneously injected with (a) 5 mL/kg of vehicle,three times a week, on days 0, 7, and 14 (G 01); (b) 5 mg/kg of ASO 1 ona weekly basis, on days 0, 7, and 14 (G 20); (c) a single dose of 5mg/kg of ds-siNA-0147 on day 0 (G 24); or (d) a combination of ASO 1 andds-siNA-0147, wherein ASO 1 was administered at a dose of 5 mg/kg on aweekly basis, on days 0, 7, and 14; and ds-siNA-0160 was administered asa single dose of 5 mg/kg at day 0 (G25). Serial blood collections wereusually taken every 5 days on day 0, 5, 10, and 15, etc. until thetermination of the study. Serum HBV S antigen (HBsAg) was assayedthrough ELISA. FIG. 16 shows a graph of the change in serum HBsAg fromAAV-HBV mice treated with vehicle (G 01, circle), ASO 1 (G 20, square),ds-siNA-0147 (G 24, diamond), or a combination of ds-siNA-0147 and ASO 1(G 25, triangle). As shown in FIG. 16 , treatment with ASO 1,ds-siNA-0147, or a combination of ASO 1 and ds-siNA-0147 resulted in areduction in serum, with the greatest reduction observed in mice treatedwith the combination of ASO 1 and ds-siNA-0147.

ds-siNA SEQ ID Strand Sequence ID NO: ds-siNA- Sense5′-mGpsmUpsmGmGfUmGfGfAf 438 0147 CmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc4-3′ Anti- 3′-mApsmGpsmCmAmCfCmAfCm 501 senseCmUmGmAmAmGmAfGmAmGmUpsf UpsmA-5′

Example 58. Efficacy of a Combination Therapy in AAV-HBV Mouse Model

This example investigates the efficacy of a combination therapycomprising an antisense oligonucleotide (ASO 1, 5′GalNAc4-ps-GalNAc4-ps-GalNAc4-po-mA-po-lnGpslnApslnTpslnApslnApsApsAps(50H)CpsGps(5m)Cps(5 m)CpsGps(5 m)CpslnApslnG pslnApscp(5 m)C-3′(SEQ ID NO: 534)) anda ds-siNA-0109 for treating HBV in an AAV-HBV mouse model.

AAV-HBV mice were subcutaneously injected with (a) 5 mL/kg of vehicle,three times a week, on days 0, 7, and 14 (G 01); (b) 5 mg/kg of ASO 1 ona weekly basis, on days 0, 7, and 14 (G 20); (c) a single dose of 5mg/kg of ds-siNA-0109 on day 0 (G 26); or (d) a combination of ASO 1 andds-siNA-0109, wherein ASO 1 was administered at a dose of 5 mg/kg on aweekly basis, on days 0, 7, and 14; and ds-siNA-0160 was administered asa single dose of 5 mg/kg at day 0 (G27). Serial blood collections wereusually taken every 5 days on day 0, 5, 10, and 15, etc. until thetermination of the study. Serum HBV S antigen (HBsAg) was assayedthrough ELISA. FIG. 17 shows a graph of the change in serum HBsAg fromAAV-HBV mice treated with vehicle (G 01, circle), ASO 1 (G 20, square),ds-siNA-0109 (G 26, diamond), or a combination of ds-siNA-0109 and ASO 1(G 27, triangle). As shown in FIG. 17 , treatment with ASO 1,ds-siNA-0109, or a combination of ASO 1 and ds-siNA-0109 resulted in areduction in serum, with the greatest reduction observed in mice treatedwith the combination of ASO 1 and ds-siNA-0109.

ds-siNA SEQ ID Strand Sequence ID NO: ds-siNA- Sense5′-mCpsmCpsmGmUfGmUfGfCf 424 0109 AmCmUmUmCmGmCmUmUmCmAp- ps2-GalNAc4Antisense 3′-mCpsmUpsmGmGfCmAmCfAm 485 CmGmUmGmAfAmGmCfGmAmApsf GpsmU-5′

Example 59. Role of 2′-Fluoro Mimics on siNA Activity

This example investigates the role of 2′-fluoro mimics, f4P and f2Pmonomers, on siNA activity. The f4P monomer was produced as described inExample 42. The f2P monomer was produced as described in Example 45.

The activity of ds-siNA-0173, ds-siNA-0174, and ds-siNA-0175 was assayedusing an in vitro HBsAg secretion assay with HepG2.2.15 cells.Generally, HepG2.2.15 cells were maintained in DMEM medium with 10%fetal bovine serum (FBS) and 1% penicillin/streptomycin, 1% Glutamine,1% non-essential amino acids, 1% Sodium Pyruvate and 250 ug/ml G418.Cells were maintained at 37° C. in a 5% CO² atmosphere. For HBsAgrelease assay, an assay medium was made that DMEM with 5% FBS, 1%penicillin/streptomycin, 1% Glutamine and 1% DMSO. The day before theassay, HepG2.2.15 cells were trypsinized and washed with Assay Mediumonce, then spun at 250 g×5 min, resuspended with Assay Medium. Theresuspenced cells were seeded at 50,000/well in assay medium in collagencoated 96 well plates. On the next day, siRNA was diluted with Opti-MEM,9-pt, 3-fold dilution and dilute Lipofectamine RNAiMAX (Invitrogen)according manufacturer's manual. siRNA dilution and RNAiMAX dilutionwere mixed and incubated at room temperature for 5 minutes. 15 μl of thesiRNA/RNAiMax mixture was added each well of the collagen coated 96 wellplate. The plates were placed in a 37° C., 5% CO² incubator for 4 days.After incubation, the supernatant was harvested and measured for HBsAgwith ELISA kit (Diasino). The cell viability was measured withCellTiter-Glo (Promega). The EC50, the concentration of the drugrequired for reducing HBsAg secretion by 50% in relation to theuntreated cell control, was calculated using the Prism Graphpad. TheCC50, the concentration of the drug required for reducing cell viabilityby 50% in relation to the untreated cell control, was calculated withthe same software. The EC50 and CC50 values are shown in Table 11.

TABLE 11 siNA Activity ds-siNA SEQ ID EC50 CC50 ID Strand Sequence NO:(nM)* (nM) ds-siNA- Sense 5′-mGpsmUpsmGmGfUmGfGfAfC 438 C >1 0173mUmUmCmUmCmUmCmAmAmU Antisense 3′-mApsmGpsmCmAmCfCmA 537fCmCmUmGmAmAmGmAfGmAmG mUpsf4PpsmA-5′ ds-siNA- Sense5′-mGpsmUpsmGmGfUmGfGfAfC 438 A >1 0174 mUmUmCmUmCmUmCmAmAmU Antisense3′-mApsmGpsmCmAmCfCmAf2P 538 mCmUmGmAmAmGmAfGmAmGm UpsfUpsmA-5′ ds-siNA-Sense 5′-mGpsmUpsmGmGfUmGfGfAfC 438 B >1 0175 mUmUmCmUmCmUmCmAmAmU(control) Antisense 5′-mApsfUpsmUmGmAfGmAmG 501 mAmAmGmUmCfCmAfCmCmAmCpsmGpsmA-3′ *A = EC50 < 0.2 nM; B = 0.2 nM < EC50 < 0.1 nm; C = EC50 >0.1 nm f4P =

f2P =

Example 60. Role of 2′-Fluoro Mimics on siNA Activity

This example investigates the role of 2′-fluoro mimics, f4P, f2P, and fxmonomers, on siNA activity of Ga1NAc4 conjugated siNAs. The f4P monomerwas produced as described in Example 42. The f2P monomer was produced asdescribed in Example 45. The fx monomer was produced as described inExample 41.

ds-siNA SEQ ID ID Strand Sequence NO: ds-siNA- Sense5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmU 438 0176 mCmAmAmU-p-(PS)2-GalNAc4Antisense 3′-mApsmGpsmCmAmCfCmAfCmCmUmGmAmAmGmA 537 fGmAmGmUpsf4PpsmA-5′ds-siNA- Sense 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmU 438 0177mCmAmAmU-p-(PS)2-GalNAc4 Antisense 3′-mApsmGpsmCmAmCfCmAf2PmCmUmGmAmAmGm538 AfGmAmGmUpsfUpsmA-5′ ds-siNA- Sense5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmU 438 0178 mCmAmAmU-p-(PS)2-GalNAc4Antisense 3′-mApsmGpsmCmAmCfCmAfXmCmUmGmAmAmG 539 mAfGmAmGmUpsfUpsmA-5′ds-siNA- Sense 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmU 438 0147mCmAmAmU-p-ps2-GalNAc4 Antisense 3′-mApsmGpsmCmAmCfCmAfCmCmUmGmAmAmG 501mAfGmAmGmUpsfUpsmA-5′ f4P =

f2P =

fX =

The activity of ds-siNA-017, ds-siNA-017, ds-siNA-017, and ds-siNA-0147can be assayed using in vitro or in vivo methods. An exemplary in vitroassay can be performed as follows:

Homo sapiens HepG2.2.15 cells are cultured in Dulbecco's ModifiedEagle's Medium (DMEM) (ATCC 30-2002) supplemented to also contain 10%fetal calf serum (FCS). Cells were incubated at 37° C. in an atmospherewith 5% CO₂ in a humidified incubator. For transfection of HepG2.2.15cells with HBV targeting siRNAs, cells are seeded at a density of 15000cells/well in 96-well regular tissue culture plates. Transfection ofcells is carried out using RNAiMAX (Invitrogen/Life Technologies)according to the manufacturer's instructions. Dose-response experimentsare done with oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625,0.3125, 0.15625 and 0.07813 nM. For each HBV targeting siRNA treatment(e.g., ds-siNA-0176, ds-siNA-0177, ds-siNA-0178, or ds-siNA-0147), fourwells are transfected in parallel, and individual data points werecollected from each well. After 24 h of incubation with siRNA, media isremoved, and cells are lysed and analyzed with a QuantiGene2.0 branchedDNA (bDNA) probe set specific for HBV genotype D (also called HepatitisB virus subtype ayw, complete genome of 3182 base-pairs) as present incell line HepG2.2.15.

For each well, the HBV on-target mRNA levels is normalized to the GAPDHmRNA level. The activity of the HBV targeting ds-siNAs can be expressedas EC50, 50% reduction of normalized HBV RNA level from no drug control.The cytotoxicity of the HBV targeting ds-siRNAs can be expressed by CC50of 50% reduction of GAPDH mRNA from no drug control.

The AAV/HBV model can be used to evaluate the in vivo activity of thesiRNA treatment (e.g., ds-siNA-0173, ds-siNA-0174, ds-siNA-0175, andds-siNA-0147). Mice are infected with AAV-HBV on day −28 of the study.AAV-HBV mice are subcutaneously injected with a single dose of 5 mL/kgof vehicle or 5 mg/kg of ds-siNA-0173, ds-siNA-0174, ds-siNA-0175, ords-siNA-0147 on day 0. Serial blood collections can be taken every 5days on day 0, 5, 10, and 15, etc. until the termination of the study.Serum HBV S antigen (HBsAg) can be assayed through ELISA.

Exemplary Embodiments

Exemplary Embodiments are Provided Below:

1. A short interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a sense strand comprising a first nucleotide sequence that        is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or        100% identical to an RNA corresponding to a target gene, wherein        the first nucleotide sequence:        -   (i) is 15 to 30 nucleotides in length; and        -   (ii) comprises 15 or more modified nucleotides independently            selected from a 2′-O-methyl nucleotide and a 2′-fluoro            nucleotide, wherein at least one modified nucleotide is a            2′-O-methyl nucleotide and the nucleotide at position 3, 5,            7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end of            the first nucleotide sequence is a 2′-fluoro nucleotide; and    -   (b) an antisense strand comprising a second nucleotide sequence        that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,        or 100% complementary to the RNA corresponding to the target        gene, wherein the second nucleotide sequence:        -   (i) is 15 to 30 nucleotides in length; and        -   (ii) comprises 15 or more modified nucleotides independently            selected from a 2′-O-methyl nucleotide and a 2′-fluoro            nucleotide, wherein at least one modified nucleotide is a            2′-O-methyl nucleotide and at least one modified nucleotide            is a 2′-fluoro nucleotide.

2. A short interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a sense strand comprising a first nucleotide sequence that        is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or        100% identical to an RNA corresponding to a target gene, wherein        the first nucleotide sequence:        -   (i) is 15 to 30 nucleotides in length; and        -   (ii) comprises 15 or more modified nucleotides independently            selected from a 2′-O-methyl nucleotide and a 2′-fluoro            nucleotide, wherein at least one modified nucleotide is a            2′-O-methyl nucleotide and at least one modified nucleotide            is a 2′-fluoro nucleotide; and    -   (b) an antisense strand comprising a second nucleotide sequence        that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,        or 100% complementary to the RNA corresponding to the target        gene, wherein the second nucleotide sequence:        -   (i) is 15 to 30 nucleotides in length; and        -   (ii) comprises 15 or more modified nucleotides independently            selected from a 2′-O-methyl nucleotide and a 2′-fluoro            nucleotide, wherein at least one modified nucleotide is a            2′-O-methyl nucleotide and the nucleotide at position 2, 5,            6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of the            second nucleotide sequence is a 2′-fluoro nucleotide.

3. The siNA of embodiment 1 or 2, wherein the first nucleotide sequencecomprises 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotidesindependently selected from a 2′-O-methyl nucleotide and a 2′-fluoronucleotide.

4. The siNA of embodiment 1 or 2, wherein 70%, 75%, 80%, 85%, 90%, 95%or 100% of the nucleotides in the first nucleotide sequence are modifiednucleotides independently selected from a 2′-O-methyl nucleotide and a2′-fluoro nucleotide.

5. The siNA of any one of embodiments 1-4, wherein at least 2, 3, 4, 5,or 6 modified nucleotides of the first nucleotide sequence are 2′-fluoronucleotides.

6. The siNA of any one of embodiments 1-5, wherein no more than 10, 9,8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotidesequence are 2′-fluoro nucleotides.

7. The siNA of any one of embodiments 1-6, wherein at least 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified nucleotides of thefirst nucleotide sequence are 2′-O-methyl nucleotides.

8. The siNA of any one of embodiments 1-7, wherein no more than 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, or 2 modified nucleotides of the first nucleotide sequence are2′-O-methyl nucleotides.

9. The siRNA of any one of embodiments 1-8, wherein the secondnucleotide sequence comprises 16, 17, 18, 19, 20, 21, 22, 23, or moremodified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide.

10. The siNA of any one of embodiments 1-9, wherein 70%, 75%, 80%, 85%,90%, 95% or 100% of the nucleotides in the second nucleotide sequenceare modified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide.

11. The siNA of any one of embodiments 1-10, wherein at least 2, 3, 4,5, or 6 modified nucleotides of the second nucleotide sequence are2′-fluoro nucleotides.

12. The siNA of any one of embodiments 1-11, wherein less than or equalto 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the secondnucleotide sequence are 2′-fluoro nucleotides.

13. The siNA of any one of embodiments 1-12, wherein at least 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified nucleotides ofthe second nucleotide sequence are 2′-O-methyl nucleotides.

14. The siNA of any one of embodiments 1-12, wherein less than or equalto 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, or 2 modified nucleotides of the second nucleotidesequence are 2′-O-methyl nucleotides.

15. A short interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a sense strand comprising a first nucleotide sequence that        is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or        100% identical to an RNA corresponding to a target gene, wherein        the first nucleotide sequence:        -   (i) is 15 to 30 nucleotides in length;        -   (ii) comprises 15 or more modified nucleotides independently            selected from a 2′-O-methyl nucleotide and a 2′-fluoro            nucleotide, wherein at least one modified nucleotide is a            2′-O-methyl nucleotide and at least one modified nucleotide            is a 2′-fluoro nucleotide; and        -   (iii) comprises 1 or more phosphorothioate internucleoside            linkage; and    -   (b) an antisense strand comprising a second nucleotide sequence        that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,        or 100% complementary to the RNA corresponding to the target        gene, wherein the second nucleotide sequence:        -   (i) is 15 to 30 nucleotides in length;        -   (ii) comprises 15 or more modified nucleotides independently            selected from a 2′-O-methyl nucleotide and a 2′-fluoro            nucleotide, wherein at least one modified nucleotide is a            2′-O-methyl nucleotide and at least one modified nucleotide            is a 2′-fluoro nucleotide; and        -   (iii) comprises 1 or more phosphorothioate internucleoside            linkage.

16. A short interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a sense strand comprising a first nucleotide sequence that        is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or        100% identical to an RNA corresponding to a target gene, wherein        the first nucleotide sequence:        -   (i) is 15 to 30 nucleotides in length; and        -   (ii) comprises 15 or more modified nucleotides independently            selected from a 2′-O-methyl nucleotide and a 2′-fluoro            nucleotide, wherein at least one modified nucleotide is a            2′-O-methyl nucleotide and at least one modified nucleotide            is a 2′-fluoro nucleotide; and    -   (b) an antisense strand comprising a second nucleotide sequence        that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,        or 100% complementary to the RNA corresponding to the target        gene, wherein the second nucleotide sequence:        -   (i) is 15 to 30 nucleotides in length; and        -   (ii) comprises 15 or more modified nucleotides independently            selected from a 2′-O-methyl nucleotide and a 2′-fluoro            nucleotide, wherein at least one modified nucleotide is a            2′-O-methyl nucleotide and at least one modified nucleotide            is a 2′-fluoro nucleotide,

wherein the siNA further comprises a phosphorylation blocker, agalactosamine, or 5′-stabilized end cap.

17. The siNA according to any preceding embodiment, wherein at least 1,2, 3, 4, 5, 6, or 7 nucleotides at position 3, 5, 7, 8, 9, 10, 11, 12,and/or 17 from the 5′ end of the first nucleotide sequence is a2′-fluoro nucleotide.

18. The siNA according to any preceding embodiment, wherein thenucleotide at position 3 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

19. The siNA according to any preceding embodiment, wherein thenucleotide at position 5 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

20. The siNA according to any preceding embodiment, wherein thenucleotide at position 7 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

21. The siNA according to any preceding embodiment, wherein thenucleotide at position 8 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

22. The siNA according to any preceding embodiment, wherein thenucleotide at position 9 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

23. The siNA according to any preceding embodiment, wherein thenucleotide at position 12 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

24. The siNA according to any preceding embodiment, wherein thenucleotide at position 17 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

25. The siNA according to any preceding embodiment, wherein thenucleotide at position 10 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

26. The siNA according to any preceding embodiment, wherein thenucleotide at position 11 from the 5′ end of the first nucleotidesequence is a 2′-fluoro nucleotide.

27. The siNA according to any preceding embodiment, wherein at least 1,2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at position 2, 5, 6, 8, 10, 14,16, 17, and/or 18 from the 5′ end of the second nucleotide sequence is a2′-fluoro nucleotide.

28. The siNA according to any preceding embodiment, wherein thenucleotide at position 2 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

29. The siNA according to any preceding embodiment, wherein thenucleotide at position 5 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

30. The siNA according to any preceding embodiment, wherein thenucleotide at position 6 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

31. The siNA according to any preceding embodiment, wherein thenucleotide at position 8 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

32. The siNA according to any preceding embodiment, wherein thenucleotide at position 10 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

33. The siNA according to any preceding embodiment, wherein thenucleotide at position 14 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

34. The siNA according to any preceding embodiment, wherein thenucleotides at position 16 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

35. The siNA according to any preceding embodiment, wherein thenucleotide at position 17 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

36. The siNA according to any preceding embodiment, wherein thenucleotide at position 18 from the 5′ end of the second nucleotidesequence is a 2′-fluoro nucleotide.

37. The siNA according to any preceding embodiment, wherein thenucleotides in the second nucleotide sequence are arranged in analternating 1:3 modification pattern, and wherein 1 nucleotide is a2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides.

38. The siNA of embodiment 37, wherein the alternating 1:3 modificationpattern occurs 2-5 times.

39. The siNA according to embodiment 37 or 38, wherein at least two ofthe alternating 1:3 modification pattern occur consecutively.

40. The siNA according to any of embodiments 37-39, wherein at least twoof the alternating 1:3 modification pattern occurs nonconsecutively.

41. The siNA according to any of clams 37-40, wherein at least 1, 2, 3,4, or 5 alternating 1:3 modification pattern begins at nucleotideposition 2, 6, 10, 14, and/or 18 from the 5′ end of the antisensestrand.

42. The siNA according to any of clams 37-41, wherein at least onealternating 1:3 modification pattern begins at nucleotide position 2from the 5′ end of the antisense strand.

43. The siNA according to any of clams 37-42, wherein at least onealternating 1:3 modification pattern begins at nucleotide position 6from the 5′ end of the antisense strand.

44. The siNA according to any of clams 37-43, wherein at least onealternating 1:3 modification pattern begins at nucleotide position 10from the 5′ end of the antisense strand.

45. The siNA according to any of clams 37-44, wherein at least onealternating 1:3 modification pattern begins at nucleotide position 14from the 5′ end of the antisense strand.

46. The siNA according to any of clams 37-45, wherein at least onealternating 1:3 modification pattern begins at nucleotide position 18from the 5′ end of the antisense strand.

47. The siNA according to any one of embodiments 1-37, wherein thenucleotides in the second nucleotide sequence are arranged in analternating 1:2 modification pattern, and wherein 1 nucleotide is a2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl nucleotides.

48. The siNA of embodiment 47, wherein the alternating 1:2 modificationpattern occurs 2-5 times.

49. The siNA according to embodiment 47 or 48, wherein at least two ofthe alternating 1:2 modification pattern occurs consecutively.

50. The siNA according to any of embodiments 47-49, wherein at least twoof the alternating 1:2 modification pattern occurs nonconsecutively.

51. The siNA according to any of clams 47-50, wherein at least 1, 2, 3,4, or 5 alternating 1:2 modification pattern begins at nucleotideposition 2, 5, 8, 14, and/or 17 from the 5′ end of the antisense strand.

52. The siNA according to any of clams 47-51, wherein at least onealternating 1:2 modification pattern begins at nucleotide position 2from the 5′ end of the antisense strand.

53. The siNA according to any of clams 47-52, wherein at least onealternating 1:2 modification pattern begins at nucleotide position 5from the 5′ end of the antisense strand.

54. The siNA according to any of clams 47-53, wherein at least onealternating 1:2 modification pattern begins at nucleotide position 8from the 5′ end of the antisense strand.

55. The siNA according to any of clams 47-54, wherein at least onealternating 1:2 modification pattern begins at nucleotide position 14from the 5′ end of the antisense strand.

56. The siNA according to any of clams 47-55, wherein at least onealternating 1:2 modification pattern begins at nucleotide position 17from the 5′ end of the antisense strand.

57. A short interfering nucleic acid (siNA) molecule represented byFormula (VIII): 5′-An¹Bn²An³Bn⁴An⁵Bn⁶An⁷Bn⁸An⁹-3′3′-Cq¹Aq²Bq³Aq⁴Bq⁵Aq⁶Bq⁷Aq⁸Bq⁹Aq¹⁰Bq¹¹Aq¹²-5′ wherein:

-   -   the top strand is a sense strand comprising a first nucleotide        sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,        90%, 95%, or 100% identical to an RNA corresponding to a target        gene, wherein the first nucleotide sequence comprises 15 to 30        nucleotides;    -   the bottom strand is an antisense strand comprising a second        nucleotide sequence that is at least about 60%, 65%, 70%, 75%,        80%, 85%, 90%, 95%, or 100% complementary to the RNA        corresponding to the target gene, wherein the second nucleotide        sequence comprises 15 to 30 nucleotides;    -   each A is independently a 2′-O-methyl nucleotide or a nucleotide        comprising a 5′-stabilized end cap or a phosphorylation blocker;    -   B is a 2′-fluoro nucleotide;    -   C represents overhanging nucleotides and is a 2′-O-methyl        nucleotide;    -   n¹=1-4 nucleotides in length;    -   each n², n⁶, n⁸, q³, q⁵, q⁷, q⁹, q¹¹, and q¹² is independently        0-1 nucleotides in length;    -   each n³ and n⁴ is independently 1-3 nucleotides in length;    -   n⁵ is 1-10 nucleotides in length;    -   n⁷ is 0-4 nucleotides in length;    -   each n⁹, q¹, and q² is independently 0-2 nucleotides in length;    -   q⁴ is 0-3 nucleotides in length;    -   q⁶ is 0-5 nucleotides in length;    -   q⁸ is 2-7 nucleotides in length; and    -   q¹⁰ is 2-11 nucleotides in length.

58. A short interfering nucleic acid (siNA) molecule represented byFormula (IX):

5′-A₂₋₄B₁A₁₋₃B₂₋₃ A₂₋₁₀B₀₋₁A₀₋₄B₀₋₁A₀₋₂-3′3′-C₂A₀₋₂B₀₋₁A₀₋₃B₀₋₁A₀₋₅B₀₋₁A₂₋₇B₁A₂₋₁₁B₁A₁-5′ wherein:

-   -   the top strand is a sense strand comprising a first nucleotide        sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,        90%, 95%, or 100% identical to an RNA corresponding to a target        gene, wherein the first nucleotide sequence comprises 15 to 30        nucleotides;    -   the bottom strand is an antisense strand comprising a second        nucleotide sequence that is at least about 60%, 65%, 70%, 75%,        80%, 85%, 90%, 95%, or 100% complementary to the RNA        corresponding to the target gene, wherein the second nucleotide        sequence comprises 15 to 30 nucleotides;    -   each A is independently a 2′-O-methyl nucleotide or a nucleotide        comprising a 5′-stabilized end cap or a phosphorylation blocker;    -   B is a 2′-fluoro nucleotide;    -   C represents overhanging nucleotides and is a 2′-O-methyl        nucleotide.

59. A short interfering nucleic acid (siNA) molecule comprising

-   -   (a) a sense strand comprising a first nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end        of the first nucleotide sequence, and wherein 2′-O-methyl        nucleotides are at positions 1, 2, 4-6, 10, 11, and 13-16 from        the 5′ end of the first nucleotide sequence; and    -   (b) an antisense strand comprising a second nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 2 and 14 from the 5′ end of the        second nucleotide sequence, and wherein 2′-O-methyl nucleotides        are at positions 1, 3-13, and 15-17 from the 5′ end of the        second nucleotide sequence.

60. The siNA molecule of embodiment 59, wherein the first nucleotidesequence consists of 19 nucleotides.

61. The siNA molecule of embodiment 60, wherein 2′-O-methyl nucleotidesare at positions 18 and 19 from the 5′ end of the first nucleotidesequence.

62. The siNA molecule according to any one of embodiments 59-61, whereinthe second nucleotide sequence consists of 21 nucleotides.

63. The siNA molecule of embodiment 62, wherein 2′-O-methyl nucleotidesare at positions 18-21 from the 5′ end of the second nucleotidesequence.

64. A short interfering nucleic acid (siNA) molecule comprising

-   -   (a) a sense strand comprising a first nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 3, 7, 8, and 17 from the 5′ end of        the first nucleotide sequence, and wherein 2′-O-methyl        nucleotides are at positions 1, 2, 4-6, and 9-16 from the 5′ end        of the first nucleotide sequence; and    -   (b) an antisense strand comprising a second nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 2 and 14 from the 5′ end of the        first nucleotide sequence; and wherein 2′-O-methyl nucleotides        are at positions 1, 3-13, and 15-17 from the 5′ end of the first        nucleotide sequence.

65. The siNA molecule of embodiment 64, wherein the first nucleotidesequence consists of 19 nucleotides.

66. The siNA molecule of embodiment 65, wherein 2′-O-methyl nucleotidesare at positions 18 and 19 from the 5′ end of the first nucleotidesequence.

67. The siNA molecule according to any one of embodiments 64-66, whereinthe second nucleotide sequence consists of 21 nucleotides.

68. The siNA molecule of embodiment 67, wherein 2′-O-methyl nucleotidesare at positions 18-21 from the 5′ end of the second nucleotidesequence.

69. A short interfering nucleic acid (siNA) molecule comprising

-   -   (a) a sense strand comprising a first nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 3, 7-9, 12 and 17 from the 5′ end        of the first nucleotide sequence, and wherein 2′-O-methyl        nucleotides are at positions 1, 2, 4-6, 10, 11, and 13-16 from        the 5′ end of the first nucleotide sequence; and    -   (b) an antisense strand comprising a second nucleotide sequence        consisting of 17 to 23 nucleotides, wherein the nucleotides in        the second nucleotide sequence are arranged in an alternating        1:3 modification pattern, and wherein 1 nucleotide is a        2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl        nucleotides.

70. The siNA molecule of embodiment 69, wherein the first nucleotidesequence consists of 19 nucleotides.

71. The siNA molecule of embodiment 70, wherein 2′-O-methyl nucleotidesare at positions 18 and 19 from the 5′ end of the first nucleotidesequence.

72. The siNA molecule according to any one of embodiments 69-71, whereinthe second nucleotide sequence consists of 21 nucleotides.

73. The siNA molecule of embodiment 72, wherein 2′-O-methyl nucleotidesare at positions 19-21 from the 5′ end of the second nucleotidesequence.

74. The siRNA molecule according to any one of embodiments 69-73,wherein the alternating 1:3 modification pattern occurs 2-5 times.

75. The siRNA molecule according to any one of embodiments 69-74,wherein at least two of the alternating 1:3 modification pattern occurconsecutively.

76. The siRNA molecule according to any one of embodiments 69-75,wherein at least two of the alternating 1:3 modification pattern occursnonconsecutively.

77. The siNA according to any one of embodiments 69-76, wherein at least1, 2, 3, 4, or 5 alternating 1:3 modification pattern begins atnucleotide position 2, 6, 10, 14, and/or 18 from the 5′ end of theantisense strand.

78. The siNA according to any one of embodiments 69-77, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position 2from the 5′ end of the antisense strand.

79. The siNA according to any one of embodiments 69-78, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position 6from the 5′ end of the antisense strand.

80. The siNA according to any one of embodiments 69-79, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position10 from the 5′ end of the antisense strand.

81. The siNA according to any one of embodiments 69-80, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position14 from the 5′ end of the antisense strand.

82. The siNA according to any one of embodiments 69-81, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position18 from the 5′ end of the antisense strand.

83. A short interfering nucleic acid (siNA) molecule comprising

-   -   (a) a sense strand comprising a first nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 5 and 7-9 from the 5′ end of the        first nucleotide sequence, and wherein 2′-O-methyl nucleotides        are at positions 1-4, 6, and 10-17 from the 5′ end of the first        nucleotide sequence; and    -   (b) an antisense strand comprising a second nucleotide sequence        consisting of 17 to 23 nucleotides, wherein the nucleotides in        the second nucleotide sequence are arranged in an alternating        1:3 modification pattern, and wherein 1 nucleotide is a        2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl        nucleotides.

84. The siNA molecule of embodiment 83, wherein the first nucleotidesequence consists of 19 nucleotides.

85. The siNA molecule of embodiment 84, wherein 2′-O-methyl nucleotidesare at positions 18 and 19 from the 5′ end of the first nucleotidesequence.

86. The siNA molecule according to any one of embodiments 83-85, whereinthe second nucleotide sequence consists of 21 nucleotides.

87. The siNA molecule of embodiment 86, wherein 2′-O-methyl nucleotidesare at positions 19-21 from the 5′ end of the second nucleotidesequence.

88. The siRNA molecule according to any one of embodiments 83-87,wherein the alternating 1:3 modification pattern occurs 2-5 times.

89. The siRNA molecule according to any one of embodiments 83-88,wherein at least two of the alternating 1:3 modification pattern occurconsecutively.

90. The siRNA molecule according to any one of embodiments 83-89,wherein at least two of the alternating 1:3 modification pattern occursnonconsecutively.

91. The siNA according to any one of embodiments 83-90, wherein at least1, 2, 3, 4, or 5 alternating 1:3 modification pattern begins atnucleotide position 2, 6, 10, 14, and/or 18 from the 5′ end of theantisense strand.

92. The siNA according to any one of embodiments 83-91, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position 2from the 5′ end of the antisense strand.

93. The siNA according to any one of embodiments 83-92, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position 6from the 5′ end of the antisense strand.

94. The siNA according to any one of embodiments 83-93, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position10 from the 5′ end of the antisense strand.

95. The siNA according to any one of embodiments 83-94, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position14 from the 5′ end of the antisense strand.

96. The siNA according to any one of embodiments 83-95, wherein at leastone alternating 1:3 modification pattern begins at nucleotide position18 from the 5′ end of the antisense strand.

97. A short interfering nucleic acid (siNA) molecule comprising

-   -   (a) a sense strand comprising a first nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 5 and 7-9 from the 5′ end of the        first nucleotide sequence, and wherein 2′-O-methyl nucleotides        are at positions 1-4, 6, and 10-17 from the 5′ end of the first        nucleotide sequence; and    -   (b) an antisense strand comprising a second nucleotide sequence        consisting of 17 to 23 nucleotides, wherein the nucleotides in        the second nucleotide sequence are arranged in an alternating        1:2 modification pattern, and wherein 1 nucleotide is a        2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl        nucleotides.

98. The siNA molecule of embodiment 97, wherein the first nucleotidesequence consists of 19 nucleotides.

99. The siNA molecule of embodiment 98, wherein 2′-O-methyl nucleotidesare at positions 18 and 19 from the 5′ end of the first nucleotidesequence.

100. The siNA molecule according to any one of embodiments 97-99,wherein the second nucleotide sequence consists of 21 nucleotides.

101. The siNA molecule of embodiment 100, wherein 2′-O-methylnucleotides are at positions 18-21 from the 5′ end of the secondnucleotide sequence.

102. The siRNA molecule according to any one of embodiments 97-101,wherein the alternating 1:2 modification pattern occurs 2-5 times.

103. The siRNA molecule according to any one of embodiments 97-102,wherein at least two of the alternating 1:2 modification pattern occurconsecutively.

104. The siRNA molecule according to any one of embodiments 97-103,wherein at least two of the alternating 1:2 modification pattern occursnonconsecutively.

105. The siNA according to any one of embodiments 97-104, wherein atleast 1, 2, 3, 4, or 5 alternating 1:2 modification pattern begins atnucleotide position 2, 5, 8, 14, and/or 17 from the 5′ end of theantisense strand.

106. The siNA according to any one of embodiments 97-105, wherein atleast one alternating 1:2 modification pattern begins at nucleotideposition 2 from the 5′ end of the antisense strand.

107. The siNA according to any one of embodiments 97-106, wherein atleast one alternating 1:2 modification pattern begins at nucleotideposition 5 from the 5′ end of the antisense strand.

108. The siNA according to any one of embodiments 97-107, wherein atleast one alternating 1:2 modification pattern begins at nucleotideposition 8 from the 5′ end of the antisense strand.

109. The siNA according to any one of embodiments 74-85, wherein atleast one alternating 1:2 modification pattern begins at nucleotideposition 14 from the 5′ end of the antisense strand.

110. The siNA according to any one of embodiments 97-109, wherein atleast one alternating 1:2 modification pattern begins at nucleotideposition 17 from the 5′ end of the antisense strand.

111. A short interfering nucleic acid (siNA) molecule comprising

-   -   (a) a sense strand comprising a first nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 5 and 7-9 from the 5′ end of the        first nucleotide sequence, and wherein 2′-O-methyl nucleotides        are at positions 1-4, 6, and 10-17 from the 5′ end of the first        nucleotide sequence; and    -   (b) an antisense strand comprising a second nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of        the second nucleotide sequence, and wherein 2′-O-methyl        nucleotides are at positions 1, 3-5, 7-13, 15, and 17 from the        5′ end the second nucleotide sequence.

112. The siNA molecule of embodiment 111, wherein the first nucleotidesequence consists of 19 nucleotides.

113. The siNA molecule of embodiment 112, wherein 2′-O-methylnucleotides are at positions 18 and 19 from the 5′ end of the firstnucleotide sequence.

114. The siNA molecule according to any one of embodiments 111-113,wherein the second nucleotide sequence consists of 21 nucleotides.

115. The siNA molecule of embodiment 114, wherein 2′-O-methylnucleotides are at positions 18-21 from the 5′ end of the secondnucleotide sequence.

116. A short interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a sense strand comprising a first nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 5, 9-11, and 14 from the 5′ end of        the first nucleotide sequence, and wherein 2′-O-methyl        nucleotides are at positions 1-4, 6-8, and 12-17 from the 5′ end        of the first nucleotide sequence; and    -   (b) an antisense strand comprising a second nucleotide sequence        consisting of 17 to 23 nucleotides, wherein 2′-fluoro        nucleotides are at positions 2 and 14 from the 5′ end of the        second nucleotide sequence, and wherein 2′-O-methyl nucleotides        are at positions 1, 3-13, and 15-17 from the 5′ end the second        nucleotide sequence.

117. The siNA molecule of embodiment 116, wherein the first nucleotidesequence consists of 21 nucleotides.

118. The siNA molecule of embodiment 117, wherein 2′-O-methylnucleotides are at positions 18-21 from the 5′ end of the firstnucleotide sequence.

119. The siNA molecule according to any one of embodiments 116-118,wherein the second nucleotide sequence consists of 23 nucleotides.

120. The siNA molecule of embodiment 119, wherein 2′-O-methylnucleotides are at positions 18-23 from the 5′ end of the secondnucleotide sequence.

121. The siNA according to any preceding embodiment, wherein the sensestrand further comprises TT sequence adjacent to the first nucleotidesequence.

122. The siNA according to any preceding embodiment, wherein the sensestrand further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or more phosphorothioate internucleoside linkages.

123. The siNA of embodiment 122, wherein at least one phosphorothioateinternucleoside linkage is between the nucleotides at positions 1 and 2from the 5′ end of the first nucleotide sequence.

124. The siNA of embodiment 122 or 123, wherein at least onephosphorothioate internucleoside linkage is between the nucleotides atpositions 2 and 3 from the 5′ end of the first nucleotide sequence.

125. The siNA according to any preceding embodiment, wherein theantisense strand further comprises TT sequence adjacent to the secondnucleotide sequence.

126. The siNA according to any preceding embodiment, wherein theantisense strand further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more phosphorothioate internucleosidelinkages.

127. The siNA of embodiment 126, wherein at least one phosphorothioateinternucleoside linkage is between the nucleotides at positions 1 and 2from the 5′ end of the second nucleotide sequence.

128. The siNA of embodiment 126 or 127, wherein at least onephosphorothioate internucleoside linkage is between the nucleotides atpositions 2 and 3 from the 5′ end of the second nucleotide sequence.

129. The siNA of any one of embodiments 126-128, wherein at least onephosphorothioate internucleoside linkage is between the nucleotides atpositions 1 and 2 from the 3′ end of the second nucleotide sequence.

130. The siNA of any one of embodiments 126-129, wherein at least onephosphorothioate internucleoside linkage is between the nucleotides atpositions 2 and 3 from the 3′ end of the second nucleotide sequence.

131. The siNA according to any preceding embodiment, wherein the firstnucleotide from the 5′ end of the first nucleotide sequence comprises a5′ stabilizing end cap.

132. The siNA according to any preceding embodiment, wherein the firstnucleotide from the 5′ end of the second nucleotide sequence comprises a5′ stabilizing end cap.

133. The siNA according to any preceding embodiment, wherein the firstnucleotide from the 5′ end of the first nucleotide sequence comprises aphosphorylation blocker.

134. The siNA according to any preceding embodiment, wherein the firstnucleotide from the 5′ end of the second nucleotide sequence comprises aphosphorylation blocker.

135. The siNA according to any preceding embodiment, wherein the firstnucleotide sequence or second nucleotide sequence comprises at least onemodified nucleotide selected from

where R is H or alkyl (or AmNA(N-Me)) when R is alkyl);

wherein B is a nucleobase.

136. A short-interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a phosphorylation blocker of Formula (IV):

wherein

-   -   R¹ is a nucleobase,    -   R⁴ is —θ-R³⁰ or —NR³¹R³²,    -   R³⁰ is C₁-C₈ substituted or unsubstituted alkyl; and    -   R³¹ and R³² together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;        and    -   (b) a short interfering nucleic acid (siNA).

137. A short-interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a 5′-stabilized end cap of Formula (Ia):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)-Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²¹, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴,    -   R²¹ and R²² are independently hydrogen or C₁-C₆ alkyl; R²¹ and        R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4; and    -   (b) a short interfering nucleic acid (siNA).

138. A short-interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a 5′-stabilized end cap of Formula (Ib):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

-   —CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆    alkenylene)-Z and R²⁰ is hydrogen; or    -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)-Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²¹, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²⁵, —NR²³R²⁴,    -   R²¹ and R²² are independently hydrogen or C₁-C₆ alkyl; R²¹ and        R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4; and    -   (b) a short interfering nucleic acid (siNA).

139. A short-interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a 5′-stabilized end cap selected from the group consisting        of Formula (1) to Formula (15), Formula (9X) to Formula (12X),        and Formula (9Y) to Formula (12Y):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H; and

-   -   (b) a short interfering nucleic acid (siNA).

140. A short-interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a 5′-stabilized end cap selected from the group consisting        of Formulas (1A)-(15A), Formulas (9B)-(12B), Formulas        (9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and        Formulas (9BY)-(12BY):

and

-   -   (b) a short interfering nucleic acid (siNA).

141. The siNA molecule according to any one of embodiments 136-140,wherein the siNA comprises the sense strand of any one of embodiments1-135.

142. The siNA molecule according to any one of embodiments 136-141,wherein the siNA comprises the antisense strand of any one ofembodiments 1-135.

143. A short interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a sense strand comprising a first nucleotide sequence that        is at least about 60%/, 65%, 70%/, 75%, 80%/, 85%, 90%/, 95%, or        100%/identical to an RNA corresponding to a target gene, wherein        the first nucleotide sequence comprises a nucleotide sequence of        any one SEQ ID NOs: 1-56, 103-158, and 205-260; and    -   (b) an antisense strand comprising a second nucleotide sequence        that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,        or 100% complementary to the RNA corresponding to the target        gene, wherein the second nucleotide sequence comprises a        nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204,        and 261-306.

144. A interfering nucleic acid (siNA) molecule comprising:

-   -   (a) a sense strand comprising a nucleotide sequence of any one        of SEQ ID NOs: 307-362 and 415-444; and    -   (b) an antisense strand comprising a nucleotide sequence of any        one of SEQ ID NOs: 363-409, 445-533, and 536-539.

145. The siNA according to any one of embodiments 1-132, 135, and137-144, wherein the siNA further comprises a phosphorylation blocker.

146. The siNA according to any one of embodiments 16, 133, 134, and 145,wherein the phosphorylation blocker has the structure of Formula (IV):

wherein

-   -   R¹ is a nucleobase,    -   R⁴ is —O—R⁰ or —NR³¹R³², R³⁰ is C₁-C₈ substituted or        unsubstituted alkyl; and    -   R³¹ and R³² together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring.

147. The siNA of embodiment 136 or 146, wherein R⁴ is —OCH₃ or—N(CH₂CH₂)₂O.

148. The siNA according to any one of embodiments 16, 133, 134, 136, and145-147, wherein the phosphorylation blocker is attached to the 5′ endof the sense strand.

149. The siNA of embodiment 148, wherein the phosphorylation blocker isattached to the 5′ end of the sense strand via one or more linkersindependently selected from a phosphodiester linker, phosphorothioatelinker, and phosphorodithioate linker.

150. The siNA according to any one of embodiments 16, 133, 134, 136, and145-147, wherein the phosphorylation blocker is attached to the 3′ endof the sense strand.

151. The siNA of embodiment 150, wherein the phosphorylation blocker isattached to the 3′ end of the sense strand via one or more linkersindependently selected from a phosphodiester linker, phosphorothioatelinker, and phosphorodithioate linker.

152. The siNA according to any one of embodiments 16, 133, 134, 136, and145-147, wherein the phosphorylation blocker is attached to the 5′ endof the antisense strand.

153. The siNA of embodiment 152, wherein the phosphorylation blocker isattached to the 5′ end of the antisense strand via one or more linkersindependently selected from a phosphodiester linker, phosphorothioatelinker, and phosphorodithioate linker.

154. The siNA according to any one of embodiments 16, 133, 134, 136, and144-147, wherein the phosphorylation blocker is attached to the 3′ endof the antisense strand.

155. The siNA of embodiment 154, wherein the phosphorylation blocker isattached to the 3′ end of the antisense strand via one or more linkersindependently selected from a phosphodiester linker, phosphorothioatelinker, and phosphorodithioate linker.

156. The siNA according to any preceding embodiment, wherein the siNAfurther comprises a galactosamine.

157. The siNA of embodiment 16 or 156, wherein the galactosamine isN-acetylgalactosamine (GalNAc) of Formula (VII):

wherein each n is independently 1 or 2.

158. The siNA of embodiment 16 or 156, wherein the galactosamine isN-acetylgalactosamine (GalNAc) of Formula (VI):

wherein

-   -   m is 1, 2, 3, 4, or 5;    -   each n is independently 1 or 2;    -   p is 0 or 1;    -   each R is independently H;    -   each Y is independently selected from —O—P(═O)(SH)—,        —O—P(═O)(O)—, —O—P(═O)(OH)—, and —O—P(S)S—;    -   Z is H or a second protecting group;    -   either L is a linker or L and Y in combination are a linker; and    -   A is H, OH, a third protecting group, an activated group, or an        oligonucleotide.

159. The siNA of embodiment 158, wherein A is an oligonucleotide.

160. The siNA of embodiment 158, wherein A is 1-2 oligonucleotides.

161. The siNA of any one of embodiments 158-160, wherein theoligonucleotide is dTdT.

162. The siNA according to any one of embodiments 16 and 156-161,wherein the galactosamine is attached to the 3′ end of the sense strand.

163. The siNA of embodiment 162, wherein the galactosamine is attachedto the 3′ end of the sense strand via one or more linkers independentlyselected from a phosphodiester linker, phosphorothioate linker, orphosphorodithioate linker.

164. The siNA according to any one of embodiments 16 and 156-161,wherein the galactosamine is attached to the 5′ end of the sense strand.

165. The siNA of embodiment 164, wherein the galactosamine is attachedto the 5′ end of the sense strand via one or more linkers independentlyselected from a phosphodiester linker, phosphorothioate linker, orphosphorodithioate linker.

166. The siNA according to any one of embodiments 16 and 156-161,wherein the galactosamine is attached to the 3′ end of the antisensestrand.

167. The siNA of embodiment 166, wherein the galactosamine is attachedto the 3′ end of the atnisense strand via one or more linkersindependently selected from a phosphodiester linker, phosphorothioatelinker, or phosphorodithioate linker.

168. The siNA according to any one of embodiments 16 and 156-161,wherein the galactosamine is attached to the 5′ end of the antisensestrand.

169. The siNA of embodiment 168, wherein the galactosamine is attachedto the 5′ end of the atnisense strand via one or more linkersindependently selected from a phosphodiester linker, phosphorothioatelinker, or phosphorodithioate linker.

170. The siNA according to any one of embodiments 1-130, 133-136, and139-169, wherein the siNA further comprises a 5′-stabilized end cap.

171. The siNA according to any one of embodiments 16, 131, 132, and 170,wherein the 5′-stabilized end cap is a 5′ vinyl phosphonate ordeuterated 5′ vinyl phosphonate.

172. The siNA according to any one of embodiments 16, 131, 132, and 170,wherein the

-   -   5′-stabilized end cap has the structure of Formula (Ia):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²¹, —NR²³R²⁴,        or —NR²³SO₂R²⁴;    -   R²¹ and R²² either are independently hydrogen or C₁-C₆ alkyl, or        R²¹ and R²² together form an oxo group;    -   R²³ is hydrogen or C1-C6 alkyl;    -   R²⁴ is —SO₂R^(2′) or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R^(2S) is C1-C6 alkyl; and    -   m is 1, 2, 3, or 4.

173. The siNA according to any one of embodiments 131, 132, and 170,wherein the 5′-stabilized end cap has the structure of Formula (Ib):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,

R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²¹R²²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand R²⁰ is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²¹, —NR²³R²⁴,        or —NR²³SO₂R²⁴;    -   R^(2′) and R²² either are independently hydrogen or C₁-C₆ alkyl,        or R²¹ and R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R²⁵ is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4.

174. The siNA of embodiment 172 or 173, wherein R¹ is an aryl.

175. The siNA of embodiment 174, wherein the aryl is a phenyl.

176. The siNA according to any one of embodiments 16, 131, 132, and 170,wherein the 5′-stabilized end cap is selected from the group consistingof Formula (1) to Formula (15), Formula (9X) to Formula (12X), andFormula (9Y) to Formula (12Y):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H.

177. The siNA according to any one of embodiments 16, 131, 132, and 170,wherein the 5′-stabilized end cap is selected from the group consistingof Formulas (1A)-(15A), Formulas (9B)-(12B), Formulas (9AX)-(12AX),Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas (9BY)-(12BY):

178. The siNA according to any one of embodiments 131, 132, and 170,wherein the 5′-stabilized end cap has the structure of Formula (Ic):

wherein

-   -   R¹ is a nucleobase, aryl, heteroaryl, or H,    -   R² is

—CH═CD-Z, —CD=CH-Z, —CD=CD-Z, —(CR²R²)_(n)-Z, or —(C₂-C₆ alkenylene)-Zand Ra is hydrogen; or

-   -   R² and R²⁰ together form a 3- to 7-membered carbocyclic ring        substituted with —(CR²¹R²²)_(n)—    -   Z or —(C₂-C₆ alkenylene)-Z;    -   n is 1, 2, 3, or 4;    -   Z is —ONR²³R²⁴, —OP(O)OH(CH₂)_(m)CO₂R²³,        —OP(S)OH(CH₂)_(m)CO₂R²³, —P(O)(OH)₂, —P(O)(OH)(OCH₃),        —P(O)(OH)(OCD₃), —SO₂(CH₂)_(m)P(O)(OH)₂, —SO₂NR²³R²¹, —NR²³R²⁴,        or —NR²³SO₂R²⁴;    -   R²¹ and R²² either are independently hydrogen or C₁-C₆ alkyl, or        R²¹ and R²² together form an oxo group;    -   R²³ is hydrogen or C₁-C₆ alkyl;    -   R²⁴ is —SO₂R²⁵ or —C(O)R²⁵; or    -   R²³ and R²⁴ together with the nitrogen to which they are        attached form a substituted or unsubstituted heterocyclic ring;    -   R^(2S) is C₁-C₆ alkyl; and    -   m is 1, 2, 3, or 4.

179. The siNA of embodiment 178, wherein R¹ is an aryl.

180. The siNA of embodiment 179, wherein the aryl is a phenyl.

181. The siNA according to any one of embodiments 16, 131, 132, and 170,wherein the 5′-stabilized end cap is selected from the group consistingof Formula (21) to Formula (35):

wherein R¹ is a nucleobase, aryl, heteroaryl, or H.

182. The siNA according to any one of embodiments 16, 131, 132, and 170,wherein the 5′-stabilized end cap is selected from the group consistingof Formulas (21A)-(35A), Formulas (29B)-(32B), Formulas (29AX)-(32AX),Formulas (29AY)-(32AY), Formulas (29BX)-(32BX), and Formulas(29BY)-(32BY):

183. The siNA according to any one of embodiments 1-182, wherein theantisense strand comprises at least one thermally destabilizingnucleotide selected from:

184. The siNA according to any one of embodiments 1-182, wherein thesense strand comprises at least one thermally destabilizing nucleotideselected from:

185. The siNA according to any one of embodiments 1-182, wherein thefirst nucleotide sequence comprises at least one thermally destabilizingnucleotide selected from:

186. The siNA according to any one of embodiments 1-182, wherein thesecond nucleotide sequence comprises at least one thermallydestabilizing nucleotide selected from:

187. The siNA according to any one of embodiments 16, 131, 132, and170-186, wherein the 5′-stabilized end cap is attached to the 5′ end ofthe antisense strand.

188. The siNA of embodiment 187, wherein the 5′-stabilized end cap isattached to the 5′ end of the antisense strand via one or more linkersindependently selected from a phosphodiester linker, phosphorothioatelinker, or phosphorodithioate linker.

189. The siNA according to any one of embodiments 16, 131, 132, and170-186, wherein the 5′-stabilized end cap is attached to the 5′ end ofthe sense strand.

190. The siNA of embodiment 189, wherein the 5′-stabilized end cap isattached to the 5′ end of the sense strand via one or more linkersindependently selected from a phosphodiester linker, phosphorothioatelinker, or phosphorodithioate linker.

191. The siNA according to any preceding embodiment, wherein the targetgene is a viral gene.

192. The siNA of embodiment 191, wherein the viral gene is from a DNAvirus.

193. The siNA of embodiment 192, wherein the DNA virus is adouble-stranded DNA (dsDNA) virus.

194. The siNA of embodiment 193, wherein the dsDNA virus is ahepadnavirus.

195. The siNA of embodiment 194, wherein the hepadnavirus is a hepatitisB virus (HBV).

196. The siNA of embodiment 195, wherein the HBV is selected from HBVgenotypes A-J.

197. The siNA of embodiment 195 or 196, wherein the target gene isselected from the S gene or X gene of the HBV.

198. The siNA according to any one of embodiments 1-197, wherein thesecond nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides withinpositions 200-720 or 1100-1700 of SEQ ID NO: 410.

199. The siNA according to any one of embodiments 1-197, wherein thesecond nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides withinpositions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or1550-1630 of SEQ ID NO: 410.

200. The siNA according to any one of embodiments 1-197, wherein thesecond nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides withinpositions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700,1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO: 410.

201. The siNA according to any one of embodiments 1-197, wherein thesecond nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides starting atposition 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466,467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581,1583, or 1584 of SEQ ID NO: 410.

202. The siNA according to any one of embodiments 1-201, wherein thefirst nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides withinpositions 200-720 or 1100-1700 of SEQ ID NO: 410.

203. The siNA according to any one of embodiments 1-201, wherein thefirst nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides withinpositions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or1550-1630 of SEQ ID NO: 410.

204. The siNA according to any one of embodiments 1-201, wherein thefirst nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides withinpositions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700,1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO: 410.

205. The siNA according to any one of embodiments 1-201, wherein thefirst nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides starting atposition 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466,467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581,1583, or 1584 of SEQ ID NO: 410.

206. The siNA according to any preceding embodiment, wherein the firstnucleotide sequence comprises a nucleotide sequence of any one SEQ IDNOs: 1-56, 103-158, and 205-260.

207. The siNA according to any preceding embodiment, wherein the secondnucleotide sequence comprises a nucleotide sequence of any one of SEQ IDNOs: 57-102, 159-204, and 261-306.

208. The siNA according to any preceding embodiment, wherein the sensestrand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362and 415-444.

209. The siNA according to any preceding embodiment, wherein theantisense strand comprises a nucleotide sequence of any one of SEQ IDNOs: 363-409, 445-533, and 536-539.

210. The siNA according to any preceding embodiment, wherein at leastone end of the siNA is a blunt end.

211. The siNA according to any preceding embodiment, wherein at leastone end of the siNA comprises an overhang, wherein the overhangcomprises at least one nucleotide.

212. The siNA according to any one of embodiments 1-209, wherein bothends of the siNA comprise an overhang, wherein the overhang comprises atleast one nucleotide.

213. The siNA according to any preceding embodiment, wherein the siNA isselected from ds-siNA-001 to ds-siNA-0178.

214. The siNA according to any preceding embodiment, wherein at leastone 2′-fluoro nucleotide or 2′-O-methyl nucleotide is a 2′-fluoro or2-O-methyl nucleotide mimic of Formula (V):

wherein

-   -   R¹ is independently a nucleobase, aryl, heteroaryl, or H, Q¹ and        Q² are independently S or O,    -   R⁵ is independently —OCD₃, —F, or —OCH₃, and    -   R⁶ and R⁷ are independently H, D, or CD3.

215. The siNA of embodiment 214, wherein the 2′-fluoro or 2′-O-methylnucleotide mimic is a nucleotide mimic of Formula (16) -Formula (20):

wherein R¹ is a nucleobase and R² is independently F or —OCH₃.

216. The siNA according to any preceding embodiment, wherein at leastone 2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic.

217. The siNA according to embodiment 216, wherein at least one2′-fluoro nucleotide on the antisense strand or the second nucleotidesequence is a 2′-fluoro nucleotide mimic.

218. The siNA according to embodiment 216 or 217, wherein the nucleotideat position 2 from the 5′ end of the antisense strand or the secondnucleotide sequence is a 2′-fluoro nucleotide mimic.

219. The siNA according to any one of embodiments 216-218, wherein thenucleotide at position 5 from the 5′ end of the antisense strand or thesecond nucleotide sequence is a 2′-fluoro nucleotide mimic.

220. The siNA according to any one of embodiments 216-219, wherein thenucleotide at position 6 from the 5′ end of the antisense strand or thesecond nucleotide sequence is a 2′-fluoro nucleotide mimic.

221. The siNA according any one of embodiments 216-220, wherein thenucleotide at position 8 from the 5′ end of the antisense strand or thesecond nucleotide sequence is a 2′-fluoro nucleotide mimic.

222. The siNA according to any one of embodiments 216-221, wherein thenucleotide at position 10 from the 5′ end of the antisense strand or thesecond nucleotide sequence is a 2′-fluoro nucleotide mimic.

223. The siNA according to any one of embodiments 216-222, wherein thenucleotide at position 14 from the 5′ end of the antisense strand or thesecond nucleotide sequence is a 2′-fluoro nucleotide mimic.

224. The siNA according to any one of embodiments 216-223, wherein thenucleotide at position 16 from the 5′ end of the antisense strand or thesecond nucleotide sequence is a 2′-fluoro nucleotide mimic.

225. The siNA according to any one of embodiments 216-224, wherein thenucleotide at position 17 from the 5′ end of the antisense strand or thesecond nucleotide sequence is a 2′-fluoro nucleotide mimic.

226. The siNA according to any one of embodiments 216-225, wherein atleast one 2′-fluoro nucleotide on the sense strand or the firstnucleotide sequence is a 2′-fluoro nucleotide mimic.

227. The siNA according to any one of embodiments 216-226, wherein thenucleotide at position 3 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

228. The siNA according to any one of embodiments 216-227, wherein thenucleotide at position 5 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

229. The siNA according to any one of embodiments 216-228, wherein thenucleotide at position 7 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

230. The siNA according to any one of embodiments 216-229, wherein thenucleotide at position 8 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

231. The siNA according to any one of embodiments 216-230, wherein thenucleotide at position 9 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

232. The siNA according to any one of embodiments 216-231, wherein thenucleotide at position 10 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

233. The siNA according to any one of embodiments 216-232, wherein thenucleotide at position 11 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

234. The siNA according to any one of embodiments 216-233, wherein thenucleotide at position 12 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

235. The siNA according to any one of embodiments 216-234, wherein thenucleotide at position 14 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

236. The siNA according to any one of embodiments 216-235, wherein thenucleotide at position 17 from the 5′ end of the sense strand or thefirst nucleotide sequence is a 2′-fluoro nucleotide mimic.

237. The siNA according to any one of embodiments 216-236, wherein atleast 1, 2, 3, 4, 5, 6, or more 2′-fluoro nucleotide mimics is a f4Pnucleotid

238. The siNA according to any one of embodiments 216-237, wherein lessthan or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 2′-fluoro nucleotidemimics is a f4P nucleotide

239. The siNA according to any one of embodiments 216-238, wherein 1, 2,3, 4, 5, 6, or more 2′-fluoro nucleotide mimics is a f2P nucleotide

240. The siNA according to any one of embodiments 216-239, wherein lessthan or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 2′-fluoro nucleotidemimics is a f2P nucleotide

241. The siNA according to any one of embodiments 216-240, wherein 1, 2,3, 4, 5, 6, or more 2′-fluoro nucleotide mimics is a fX nucleotide

242. The siNA according to any one of embodiments 216-241, wherein lessthan or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 2′-fluoro nucleotidemimics is a fX nucleotide

243. The siNA according to any preceding embodiment, wherein the firstnucleotide from the 5′ end of the sense strand or first nucleotidesequence is a d2vd3 nucleotide

244. The siNA according to any preceding embodiment, wherein the firstnucleotide from the 3′ end of the sense strand or first nucleotidesequence is a d2vd3 nucleotide

245. The siNA according to any preceding embodiment, wherein the firstnucleotide from the 5′ end of the antisense strand or second nucleotidesequence is a d2vd3 nucleotide

246. The siNA according to any preceding embodiment, wherein the firstnucleotide from the 3′ end of the antisense strand or second nucleotidesequence is a d2vd3 nucleotide

247. A composition comprising the siNA according to any one ofembodiments 1-246.

248. A composition comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more siNAsaccording to any one of embodiments 1-246.

249. The composition of embodiment 248, wherein at least 1, 2, 3, 4, 5,or more siNAs target an S gene of HBV.

250. The composition of embodiment 248 or 249, wherein at least 1, 2, 3,4, 5, or more siNAs target an X gene of HBV.

251. The composition according to any one of embodiments 247-250,further comprising an additional HBV treatment agent.

252. The composition of embodiment 251, wherein the additional HBVtreatment agent is selected from a nucleotide analog, nucleoside analog,a capsid assembly modulator (CAM), a recombinant interferon, an entryinhibitor, a small molecule immunomodulator and oligonucleotide therapy.

253. The composition of embodiment 252, wherein the oligonucleotidetherapy is an additional siNA.

254. The composition of embodiment 253, wherein the additional siNA isselected from any of ds-siNA-001 to ds-siNA-0178.

255. The composition of embodiment 252, wherein the oligonucleotidetherapy is an antisense oligonucleotide (ASO), NAPs, or STOPS™

256. The composition of embodiment 255, wherein the ASO is ASO 1 or ASO2.

257. The composition of embodiment 251 or 252, wherein the additionalHBV treatment agent is selected from HBV STOPS™ ALG-010133, HBV CAMALG-000184, ASO 1, recombinant interferon alpha 2b, IFN-α, PEG-IFN-α-2a,lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir,tenofovir alafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109,JNJ-632, JNJ-3989 (ARO-HBV), RG6004, GSK3228836, REP-2139, REP-2165,AB-729, VIR-2218, RG6346 (DCR-HBVS), JNJ-6379, GLS4, ABI-HO731, JNJ-440,NZ-4, RG7907, EDP-514, AB-423, AB-506, ABI-H03733 and ABI-H2158.

258. A method of treating a disease in a subject in need thereof,comprising administering to the subject the siNA according to any one ofembodiments 1-246.

259. A method of treating a disease in a subject in need thereof,comprising administering to the subject the composition according to anyone of embodiments 247-257.

260. The method of embodiment 258 or 259, wherein the disease is a viraldisease.

261. The method of embodiment 260, wherein the viral disease is causedby a DNA virus.

262. The method of embodiment 261, wherein the DNA virus is a doublestranded DNA (dsDNA) virus.

263. The method of embodiment 262, wherein the dsDNA virus is ahepadnavirus.

264. The method of embodiment 263, wherein the hepadnavirus is ahepatitis B virus (HBV).

265. The method of embodiment 264, wherein the HBV is selected from HBVgenotypes A-J.

266. The method of any of embodiments 258-265, further comprisingadministering an additional HBV treatment agent.

267. The method of embodiment 266, wherein the siNA or the compositionand the additional HBV treatment agent are administered concurrently.

268. The method of embodiment 266, wherein the siNA or the compositionand the additional HBV treatment agent are administered sequentially.

269. The method of embodiment 266, wherein the siNA or the compositionis administered prior to administering the additional HBV treatmentagent.

270. The method of embodiment 266, wherein the siNA or the compositionis administered after administering the additional HBV treatment agent.

271. The method of any one of embodiments 266-270, wherein theadditional HBV treatment agent is selected from a nucleotide analog,nucleoside analog, a capsid assembly modulator (CAM), a recombinantinterferon, an entry inhibitor, a small molecule immunomodulator andoligonucleotide therapy.

272. The method of embodiment 271, wherein the oligonucleotide therapyis an additional siNA.

273. The method of embodiment 272, wherein the additional siNA isselected from any of ds-siNA-001 to ds-siNA-0178.

274. The method of embodiment 271, wherein the oligonucleotide therapyis an antisense oligonucleotide (ASO), NAPs, or STOPs.

275. The method of embodiment 274, wherein the ASO is ASO 1 or ASO 2.

276. The method of embodiment 270 or 271, wherein the additional HBVtreatment agent is selected from HBV STOPS™ ALG-010133, HBV CAMALG-000184, ASO 1, recombinant interferon alpha 2b, IFN-α, PEG-IFN-α-2a,lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir,tenofovir alafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109,JNJ-632, JNJ-3989 (ARO-HBV), RG6004, GSK3228836, REP-2139, REP-2165,AB-729, VIR-2218, RG6346 (DCR-HBVS), JNJ-6379, GLS4, ABI-HO731, JNJ-440,NZ-4, RG7907, EDP-514, AB-423, AB-506, ABI-H03733 and ABI-H2158.

277. The method of embodiment 258 or 259, wherein the disease is a liverdisease.

278. The method of embodiment 277, wherein the liver disease is anonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma(HCC).

279. The method of embodiment 278, wherein the NAFLD is nonalcoholicsteatohepatitis (NASH).

280. The method of any of embodiments 277-279 further comprisingadministering to the subject a liver disease treatment agent.

281. The method of embodiment 280, wherein the liver disease treatmentagent is selected from a peroxisome proliferator-activator receptor(PPAR) agonist, farnesoid X receptor (FXR) agonist, lipid-alteringagent, and incretin-based therapy.

282. The method of embodiment 281, wherein the PPAR agonist is selectedfrom a PPARα agonist, dual PPARα/δ agonist, PPARγ agonist, and dualPPARα/γ agonist.

283. The method of embodiment 282, wherein the dual PPARα agonist is afibrate.

284. The method of embodiment 282, wherein the PPARα/δ agonist iselafibranor.

285. The method of embodiment 282, wherein the PPARγ agonist is athiazolidinedione (TZD).

286. The method of embodiment 282, wherein TZD is pioglitazone.

287. The method of embodiment 282, wherein the dual PPARα/γ agonist issaroglitazar.

288. The method of embodiment 281, wherein the FXR agonist isobeticholic acis (OCA).

289. The method of embodiment 281, wherein the lipid-altering agent isaramchol.

290. The method of embodiment 281, wherein the incretin-based therapy isa glucagon-like peptide 1 (GLP-1) receptor agonist or dipeptidylpeptidase 4 (DPP-4) inhibitor.

291. The method of embodiment 290, wherein the GLP-1 receptor agonist isexenatide or liraglutide.

292. The method of embodiment 290, wherein the DPP-4 inhibitor issitagliptin or vildapliptin.

293. The method of any one of embodiments 280-292, wherein the siNA orcomposition and the liver disease treatment agent are administeredconcurrently.

294. The method of any one of embodiments 280-292, wherein the siNA orcomposition and the liver disease treatment agent are administeredsequentially.

295. The method of any one of embodiments 280-292, wherein the siNA orcomposition is administered prior to administering the liver diseasetreatment agent.

296. The method of any one of embodiments 280-292, wherein the siNA orcomposition is administered after administering the liver diseasetreatment agent.

297. The method of any of one embodiments 258-296, wherein the siNA orthe composition is administered at a dose of at least 1 mg/kg, 2 mg/kg,3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg,11 mg/kg, 12 mg/kg, 13 mg/kg 14 mg/kg, or 15 mg/kg.

298. The method of any of one embodiments 258-296, wherein the siNA orthe composition is administered at a dose of between 0.5 mg/kg to 50mg/kg, 0.5 mg/kg to 40 mg/kg 0.5 mg/kg to 30 mg/kg, 1 mg/kg to 50 mg/kg,1 mg/kg to 40 mg/kg, 1 mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 3 mg/kgto 50 mg/kg, 3 mg/kg to 40 mg/kg, 3 mg/kg to 30 mg/kg, 3 mg/kg to 20mg/kg, 3 mg/kg to 15 mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 50 mg/kg, 4mg/kg to 40 mg/kg, 4 mg/kg to 30 mg/kg, 4 mg/kg to 20 mg/kg, 4 mg/kg to15 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg to 40 mg/kg,5 mg/kg to 30 mg/kg, 5 mg/kg to 20 mg/kg, 5 mg/kg to 15 mg/kg, or 5mg/kg to 10 mg/kg.

299. The method of any of one embodiments 258-298, wherein the siNA orthe composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 times.

300. The method of any of one embodiments 258-298, wherein the siNA orthe composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 times a day, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a week,or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a month.

301. The method of any of one embodiments 258-300, wherein the siNA orthe composition are administered at least once every 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

302. The method of any of one embodiments 258-301, wherein the siNA orthe composition is administered for a period of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, orat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 52,53, 54, or 55 weeks.

303. The method of any of one embodiments 258-302, wherein the siNA orthe composition is administered at a single dose of 5 mg/kg.

304. The method of any of one embodiments 258-302, wherein the siNA orthe composition is administered at a single dose of 10 mg/kg.

305. The method of any of one embodiments 258-302, wherein the siNA orthe composition is administered at three doses of 10 mg/kg once a week.

306. The method of any of one embodiments 258-302, wherein the siNA orthe composition is administered at three doses of 10 mg/kg once everythree days.

307. The method of any of one embodiments 258-302, wherein the siNA orthe composition is administered at five doses of 10 mg/kg once everythree days.

308. The method of any of one embodiments 258-302, wherein the siNA orthe composition is administered at six doses of ranging from 1 mg/kg to15 mg/kg, 1 mg/kg to 10 mg/kg, 2 mg/kg to 15 mg/kg, 2 mg/kg to 10 mg/kg,3 mg/kg to 15 mg/kg, or 3 mg/kg to 10 mg/kg.

309. The method of embodiment 308, wherein the first dose and seconddose are administered at least 3 days apart.

310. The method of embodiment 308 or 309, wherein the second dose andthird dose are administered at least 4 days apart.

311. The method of any one of embodiments 308-310, wherein the thirddose and fourth dose, fourth dose and fifth dose, or fifth dose andsixth dose are administered at least 7 days apart.

312. The method of any one of embodiments 258-311, wherein the siNA orthe composition are administered in a particle or viral vector.

313. The method of embodiment 312, wherein the viral vector is a vectorof adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus,herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus,retrovirus, or rhabdovirus.

314. The method of embodiment 312, wherein the viral vector is arecombinant viral vector.

315. The method according to any one of embodiments 312-314, wherein theviral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11,AAV-12 and AAV-13.

316. The method according to any one of embodiments 258-315, wherein thesiNA or the composition is administered systemically.

317. The method according to any one of embodiments 258-315, wherein thesiNA or the composition is administered locally.

318. The method according to any one of embodiments 258-317, wherein thesiNA or the composition is administered intravenously, subcutaneously,or intramuscularly.

319. Use of the siNA according to any one of embodiments 1-246 or thecomposition according to any one of embodiments 247-257 in themanufacture of a medicament for treating a disease.

320. The use of embodiment 319, wherein the disease is a viral disease.

321. The use of embodiment 320, wherein the viral disease is caused by aDNA virus.

322. The use of embodiment 321, wherein the DNA virus is a doublestranded DNA (dsDNA virus).

323. The use of embodiment 321, wherein the dsDNA virus is ahepadnavirus.

324. The use of embodiment 323, wherein the hepadnavirus is a hepatitisB virus (HBV).

325. The use of embodiment 324, wherein the HBV is selected from HBVgenotypes A-J.

326. The use of any of one of embodiments 319-325, further comprising anadditional HBV treatment agent in the manufacture of the medicament.

327. The use of embodiment 326, wherein the additional HBV treatmentagent is selected from a nucleotide analog, nucleoside analog, a capsidassembly modulator (CAM), a recombinant interferon, an entry inhibitor,a small molecule immunomodulator and oligonucleotide therapy.

328. The use of embodiment 327, wherein the oligonucleotide therapy isan additional siNA.

329. The use of embodiment 328, wherein the additional siNA is selectedfrom any of ds-siNA-001 to ds-siNA-0178.

330. The use of embodiment 327, wherein the oligonucleotide therapy isan antisense oligonucleotide (ASO), NAPs, or STOPs.

331. The use of embodiment 330, wherein the ASO is ASO 1 or ASO2.

332. The use of embodiment 326 or 327, wherein the additional HBVtreatment agent is selected from HBV STOPS™ ALG-010133, HBV CAMALG-000184, ASO 1, recombinant interferon alpha 2b, IFN-α, PEG-IFN-α-2a,lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir,tenofovir alafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109,JNJ-632, JNJ-3989 (ARO-HBV), RG6004, GSK3228836, REP-2139, REP-2165,AB-729, VIR-2218, RG6346 (DCR-HBVS), JNJ-6379, GLS4, ABI-HO731, JNJ-440,NZ-4, RG7907, EDP-514, AB-423, AB-506, ABI-H03733 and ABI-H2158.

333. The use of embodiment 319, wherein the disease is a liver disease.

334. The use of embodiment 333, wherein the liver disease is anonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma(HCC).

335. The use of embodiment 334, wherein the NAFLD is nonalcoholicsteatohepatitis (NASH).

336. The use of any of embodiments 333-335, further comprising a liverdisease treatment agent in the manufacture of the medicament.

337. The use of embodiment 336, wherein the liver disease treatmentagent is selected from a peroxisome proliferator-activator receptor(PPAR) agonist, farnesoid X receptor (FXR) agonist, lipid-alteringagent, and incretin-based therapy.

338. The use of embodiment 337, wherein the PPAR agonist is selectedfrom a PPARα agonist, dual PPARα/δ agonist, PPARγ agonist, and dualPPARα/γ agonist.

339. The use of embodiment 338, wherein the dual PPARα agonist is afibrate.

340. The use of embodiment 338, wherein the PPARα/δ agonist iselafibranor.

341. The use of embodiment 338, wherein the PPARγ agonist is athiazolidinedione (TZD).

342. The use of embodiment 341, wherein TZD is pioglitazone.

343. The use of embodiment 338, wherein the dual PPARα/γ agonist issaroglitazar.

344. The use of embodiment 337, wherein the FXR agonist is obeticholicacis (OCA).

345. The use of embodiment 337, wherein the lipid-altering agent isaramchol.

346. The use of embodiment 337, wherein the incretin-based therapy is aglucagon-like peptide 1 (GLP-1) receptor agonist or dipeptidyl peptidase4 (DPP-4) inhibitor.

347. The use of embodiment 346, wherein the GLP-1 receptor agonist isexenatide or liraglutide.

348. The use of embodiment 346, wherein the DPP-4 inhibitor issitagliptin or vildapliptin.

349. The siNA according to any one of embodiments 1-246 for use as amedicament.

350. The composition according to any one of embodiments 247-257 for useas a medicament.

351. The siNA according to any one of embodiments 1-246 for use in thetreatment of a disease.

352. The siNA of embodiment 351, wherein the disease is a viral disease.

353. The siNA of embodiment 352, wherein the viral disease is caused bya DNA virus.

354. The siNA of embodiment 353, wherein the DNA virus is a doublestranded DNA (dsDNA virus).

355. The siNA of embodiment 354, wherein the dsDNA virus is ahepadnavirus.

356. The siNA of embodiment 355, wherein the hepadnavirus is a hepatitisB virus (HBV).

357. The siNA of embodiment 356, wherein the HBV is selected from HBVgenotypes A-J.

358. The siNA of embodiment 351, wherein the disease is a liver disease.

359. The siNA of embodiment 358, wherein the liver disease is anonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma(HCC).

360. The siNA of embodiment 359, wherein the NAFLD is nonalcoholicsteatohepatitis (NASH).

361. The composition according to any one of embodiments 247-257, foruse in the treatment of a disease.

362. The composition of embodiment 361, wherein the disease is a viraldisease.

363. The composition of embodiment 362, wherein the viral disease iscaused by a DNA virus.

364. The composition of embodiment 363, wherein the DNA virus is adouble stranded DNA (dsDNA virus).

365. The composition of embodiment 364, wherein the dsDNA virus is ahepadnavirus.

366. The composition of embodiment 365, wherein the hepadnavirus is ahepatitis B virus (HBV).

367. The composition of embodiment 366, wherein the disease is a liverdisease.

368. The composition of embodiment 367, wherein the liver disease is anonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma(HCC).

369. The composition of embodiment 368, wherein the NAFLD isnonalcoholic steatohepatitis (NASH).

TABLE 1 Non-modified Nucleotide Sequences First NucleotideSecond Nucleotide SEQ ID NO. Sequence (5′-3′) SEQ ID NO. Sequence (5′-3′ 1 ACCGUGUGCACUUCGCUUC  57 GAAGCGAAGUGCACACGGUCC  2 ACCGUGUGCACUUCGCUUC 58 GAAGCGAAGUGCACACGGU  3 ACUUCGCUUCACCUCUGCA  59 UGCAGAGGUGAAGCGAAGUGC 4 AGUGUUUGCUGACGCAACC  60 GGUUGCGUCAGCAAACACUUG  5 CAGGCGGGGUUUUUCUUGU 61 ACAAGAAAAACCCCGCCUGUA  6 CAGGCGGGGUUUUUCUUGU  62ACAAGAAAAAACCCCGCCUG  7 CAGUUUACUAGUGCCAUUU  63 AAAUGGCACUAGUAAACUGAG  8CAGUUUACUAGUGCCAUUU  64 AAAUGGCACUAGUAAACUG  9 CAUCCUGCUGCUAUGCCUC  65GAGGCAUAGCAGCAGGAUGAA 10 CAUCCUGCUGCUAUGCCUCAU  66AUGAGGCAUAGCAGCAGGAUGAA 11 CAUCCUGCUGCUAUGCCUC  67 GAGGCAUAGCAGCAGGAUG12 CCGUGUGCACUUCGCUUCA  68 UGAAGCGAAGUGCACACGGUC 13 CCGUGUGCACUUCGCUUCA 69 UGAAGCGAAGUGCACACGG 14 CCUGCUGCUAUGCCUCAUCUU  70AAGAUGAGGCAUAGCAGCAGGAU 15 CUCAGUUUACUAGUGCCAU  71 AUGGCACUAGUAAACUGAGCC16 CUCAGUUUACUAGUGCCAU  71 AUGGCACUAGUAAACUGAGCC 17 CUCAGUUUACUAGUGCCAU 71 AUGGCACUAGUAAACUGAGCC 18 CUCAGUUUACUAGUGCCAU  72 AUGGCACUAGUAAACUGAG19 CUCAGUUUACUAGUGCCAU  72 AUGGCACUAGUAAACUGAG 20 CUGCUAUGCCUCAUCUUCU 73 AGAAGAUGAGGCAUAGCAGCA 21 CUGCUAUGCCUCAUCUUCU  73AGAAGAUGAGGCAUAGCAGCA 22 CUGCUAUGCCUCAUCUUCU  74 AGAAGAUGAGGCAUAGCAG 23CUGCUAUGCCUCAUCUUCU  74 AGAAGAUGAGGCAUAGCAG 24 CUGCUGCUAUGCCUCAUCU  75AGAUGAGGCAUAGCAGCAGGA 25 CUGCUGCUAUGCCUCAUCU  76 AGAUGAGGCAUAGCAGCAG 26CUGCUGCUAUGCCUCAUCU  76 AGAUGAGGCAUAGCAGCAG 27 CUUCGCUUCACCUCUGCACGU  77ACGUGCAGAGGUGAAGCGAAGUG 28 GCACUUCGCUUCACCUCUGCA  78UGCAGAGGUGAAGCGAAGUGCAC 29 GCCGAUCCAUACUGCGGAA  79 UUCCGCAGUAUGGAUCGGCAG30 GCCGGGUUUUUCUUGUUGA  80 UUCCGCAGUAUGGAUCGGC 31 GCGGGGUUUUUCUUGUUGA 81 UCAACAAGAAAAACCCCGCCU 32 GCGGGGUUUUUCUUGUUGA  81UCAACAAGAAAAACCCCGCCU 33 GCGGGGUUUUUCUUGUUGA  82 UCAACAAGAAAAACCCCGC 34GCGGGGUUUUUCUUGUUGA  82 UCAACAAGAAAAACCCCGC 35 GCUGCUAUGCCUCAUCUUCUU  83AAGAAGAUGAGGCAUAGCAGCAG 36 GGAUGUGUCUGCGGCGUUUUA  84UAAAACGCCGCAGACACAUCCAG 37 GGCCAAAAUUCGCAGUCCC  85 GGGACUGCGAAUUUUGGCCAA38 GGCGCACCUCUCUUUACGC  86 GCGUAAAGAGAGGUGCGCCCC 39 GUAUGUUGCCCGUUUGUCC 87 GGACAAACGGGCAACAUACCU 40 GUGGUGGACUUCUCUCAAU  88AUUGAGAGAAGUCCACCACGA 41 GUGUGCACUUCGCUUCACC  89 GGUGAAGCGAAGUGCACACGG42 GUUGCCCGUUUGUCCUCUA  90 UAGAGGACAAACGGGCAACAU 43 GUUGCCCGUUUGUCCUCUA 91 UAGAGGACAAACGGGCAAC 44 UCCAUACUGCGGAACUCCU  92 AGGAGUUCCGCAGUAUGGAUC45 UCCAUACUGCGGAACUCCU  93 AGGAGUUCCGCAGUAUGGA 46 UCGUGGUGGACUUCUCUCAAU 94 AUUGAGAGAAGUCCACCACGAGU 47 UGCACUUCGCUUCACCUCU  95AGAGGUGAAGCGAAGUGCACA 48 UGCCGAUCCAUACUGCGGA  96 UCCGCAGUAUGGAUCGGCAGA49 UGCCGAUCCAUACUGCGGA  97 UCCGCAGUAUGGAUCGGCA 50 UGCUAUGCCUCAUCUUCUU 98 AAGAAGAUGAGGCAUAGCAGC 51 UGUGCACUUCGCUUCACCU  99AGGUGAAGCGAAGUGCACACG 52 UGUGCACUUCGCUUCACCU  99 AGGUGAAGCGAAGUGCACACG53 UGUGCACUUCGCUUCACCU 100 AGGUGAAGCGAAGUGCACA 54 UGUGCACUUCGCUUCACCU100 AGGUGAAGCGAAGUGCACA 55 UUGCCCGUUUGUCCUCUAA 101 UUAGAGGACAAACGGGCAACA56 UUGCCCGUUUGUCCUCUAA 102 UUAGAGGACAAACGGGCAA

TABLE 2 2′-OMe and 2′-F Modified Nucleotide Sequences First NucleotideSecond Nucleotide SEQ ID NO. Sequence (5′-3′) SEQ ID NO.Sequence (5′-3′) 103 mAmCfCmGmUmGfUfGfCmAm 159 mGfAmAmGmCmGmAmAmGmUmGmCfUmUmCmGmCfUmUmC CmAfCmAmCmGmGmUmCmC 104 mAmCfCmGmUmGfUfGfCmAm 160mGfAmAmGmCmGmAmAmGmUmGm CfUmUmCmGmCfUmUmC CmAfCmAmCmGmGmU 105mAmCfUmUmCmGfCfUfUmCm 161 mUfGmCmAmGmAmGmGmUmGmAm AfCmCmUmCmUfGmCmAAmGfCmGmAmAmGmUmGmC 106 mAmGfUmGmUmUfUfGfCmUm 162mGfGmUmUmGmCmGmUmCmAmGm GfAmCmGmCmAfAmCmC CmAfAmAmCmAmCmUmUmG 107mCmAfGmGmCmGfGfGfGmUm 163 mAfCmAmAmGmAmAmAmAmAmCm UfUmUmUmCmUfUmGmUCmCfCmGmCmCmUmGmUmA 108 mCmAfGmGmCmGfGfGfGmUm 164mAfCmAmAmGmAmAmAmAmAmAm UfUmUmUmCmUfUmGmU CmCmCfCmGmCmCmUmG 109mCmAfGmUmUmUfAfCfUmAm 165 mAfAmAmUmGmGmCmAmCmUmAm GfUmGmCmCmAfUmUmUGmUfAmAmAmCmUmGmAmG 110 mCmAfGmUmUmUfAfCfUmAm 166mAfAmAmUmGmGmCmAmCmUmAm GfUmGmCmCmAfUmUmU GmUfAmAmAmCmUmG 111mCmAfUmCmCmUfGfCfUmGm 167 mGfAmGmGmCmAmUmAmGmCmAm CfUmAmUmGmCfCmUmCGmCfAmGmGmAmUmGmAmA 112 mCmAmUmCmCmUfGmCfUfGf 168mAfUmGmAmGfGmCmAmUmAmGm CmUmAmUmGmCmCmUmCmAmU CmAfGmCfAmGmGmAmUmGmAmA113 mCmAfUmCmCmUfGfCfUmGm 169 mGfAmGmGmCmAmUmAmGmCmAm CfUmAmUmGmCfCmUmCGmCfAmGmGmAmUmG 114 mCmCfGmUmGmUfGfCfAmC 170 mUfGmAmAmGmCmGmAmAmGmUmmUfUmCmGmCmUfUmCmA GmCfAmCmAmCmGmGmUmC 115 mCmCfGmUmGmUfGfCfAmCm 171mUfGmAmAmGmCmGmAmAmGmUm UfUmCmGmCmUfUmCmA GmCfAmCmAmCmGmG 116mCmCmUmGmCmUfGmCfUfAf 172 mAfAmGmAmUfGmAmGmGmCmAm UmGmCmCmUmCmAmUmCmUmUUmAfGmCfAmGmCmAmGmGmAmU 117 mCmUfCmAmGmUfUfUfAmCm 173mAfUmGmGmCmAmCmUmAmGmUm UfAmGmUmGmCfCmAmU AmAfAmCmUmGmAmGmCmC 118mCmUmCmAmGmUfUmUmAmCm 173 mAfUmGmGmCmAmCmUmAmGmUm UmAmGmUmGmCmCmAmUAmAfAmCmUmGmAmGmCmC 119 mCmUfCmAmGmUfUfUmAmCm 173mAfUmGmGmCmAmCmUmAmGmUm UmAmGmUmGmCfCmAmU AmAfAmCmUmGmAmGmCmC 120mCmUfCmAmGmUfUfUfAmCm 174 mAfUmGmGmCmAmCmUmAmGmUm UfAmGmUmGmCfCmAmUAmAfAmCmUmGmAmG 121 mCmUfCmAmGmUfUfUmAmCm 174 mAfUmGmGmCmAmCmUmAmGmUmUmAmGmUmGmCfCmAmU AmAfAmCmUmGmAmG 122 mCmUfGmCmUmAfUfGfCmCm 175mAfGmAmAmGmAmUmGmAmGmGm UfCmAmUmCmUfUmCmU CmAfUmAmGmCmAmGmCmA 123mCmUfGmCmUmAfUfGmCmCm 175 mAfGmAmAmGmAmUmGmAmGmGm UmCmAmUmCmUfUmCmUCmAfUmAmGmCmAmGmCmA 124 mCmUfGmCmUmAfUfGfCmCm 176mAfGmAmAmGmAmUmGmAmGmGm UfCmAmUmCmUfUmCmU CmAfUmAmGmCmAmG 125mCmUfGmCmUmAfUfGmCmCm 176 mAfGmAmAmGmAmUmGmAmGmGm UmCmAmUmCmUfUmCmUCmAfUmAmGmCmAmG 126 mCmUfGmCmUmGfCfUfAmUm 177 mAfGmAmUmGmAmGmGmCmAmUmGfCmCmUmCmAfUmCmU AmGfCmAmGmCmAmGmGmA 127 mCmUfGmCmUmGfCfUfAmUm 178mAfGmAmUmGmAmGmGmCmAmUm GfCmCmUmCmAfUmCmU AmGfCmAmGmCmAmG 128mCmUfGmCmUmGfCfUmAmUm 178 mAfGmAmUmGmAmGmGmCmAmUm GmCmCmUmCmAfUmCmUAmGfCmAmGmCmAmG 129 mCmUmUmCmGmCfUmUfCfAf 179 mAfCmGmUmGfCmAmGmAmGmGmCmCmUmCmUmGmCmAmCmGmU UmGfAmAfGmCmGmAmAmGmUmG 130 mGmCmAmCmUmUfCmGfCfUf180 mUfGmCmAmGfAmGmGmUmGmAm UmCmAmCmCmUmCmUmGmCmAAmGfCmGfAmAmGmUmGmCmAmC 131 mGmCfCmGmAmUfCfCfAmU 181mUfUmCmCmGmCmAmGmUmAmUm mAfCmUmGmCmGfGmAmA GmGfAmUmCmGmGmCmAmG 132mGmCfCmGmGmGfUfUfUmUm 182 mUfUmCmCmGmCmAmGmUmAmUm UfCmUmUmGmUfUmGmAGmGfAmUmCmGmGmC 133 mGmCfGmGmGmGfUfUfUmUm 183 mUfCmAmAmCmAmAmGmAmAmAmUfCmUmUmGmUfUmGmA AmAfCmCmCmCmGmCmCmU 134 mGmCfGmGmGmGfUfUmUmUm 183mUfCmAmAmCmAmAmGmAmAmAm UmCmUmUmGmUfUmGmA AmAfCmCmCmCmGmCmCmU 135mGmCfGmGmGmGfUfUfUmUm 184 mUfCmAmAmCmAmAmGmAmAmAm UfCmUmUmGmUfUmGmAAmAfCmCmCmCmGmC 136 mGmCfGmGmGmGfUfUmUmUm 184 mUfCmAmAmCmAmAmGmAmAmAmUmCmUmUmGmUfUmGmA AmAfCmCmCmCmGmC 137 mGmCmUmGmCmUfAmUfGfCf 185mAfAmGmAmAfGmAmUmGmAmGm CmUmCmAmUmCmUmUmCmUmU GmCfAmUfAmGmCmAmGmCmAmG138 mGmGmAmUmGmUfGmUfCfUf 186 mUfAmAmAmAfCmGmCmCmGmCmGmCmGmGmCmGmUmUmUmUmA AmGfAmCfAmCmAmUmCmCmAmG 139 mGmGfCmCmAmAfAfAfUmUm187 mGfGmGmAmCmUmGmCmGmAmAm CfGmCmAmGmUfCmCmC UmUfUmUmGmGmCmCmAmA 140mGmGfCmGmCmAfCfCfUmCm 188 mGfCmGmUmAmAmAmGmAmGmAm UfCmUmUmUmAfCmGmCGmGfUmGmCmGmCmCmCmC 141 mGmUfAmUmGmUfUfGfCmCm 189mGfGmAmCmAmAmAmCmGmGmGm CfGmUmUmUmGfUmCmC CmAfAmCmAmUmAmCmCmU 142mGmUfGmGmUmGfGfAfCmUm 190 mAfUmUmGmAmGmAmGmAmAmGm UfCmUmCmUmCfAmAmUUmCfCmAmCmCmAmCmGmA 143 mGmUfGmUmGmCfAfCfUmU 191 mGfGmUmGmAmAmGmCmGmAmAmmCfGmCmUmUmCfAmCmC GmUfGmCmAmCmAmCmGmG 144 mGmUfUmGmCmCfCfGfUmU 192mUfAmGmAmGmGmAmCmAmAmAm mUfGmUmCmCmUfCmUmA CmGfGmGmCmAmAmCmAmU 145mGmUfUmGmCmCfCfGfUmUm 193 mUfAmGmAmGmGmAmCmAmAmAm UfGmUmCmCmUfCmUmACmGfGmGmCmAmAmC 146 mUmCfCmAmUmAfCfUfGmCm 194 mAfGmGmAmGmUmUmCmCmGmCmGfGmAmAmCmUfCmCmU AmGfUmAmUmGmGmAmUmC 147 mUmCfCmAmUmAfCfUfGmCm 195mAfGmGmAmGmUmUmCmCmGmCm GfGmAmAmCmUfCmCmU AmGfUmAmUmGmGmA 148mUmCmGmUmGmGfUmGfGfAf 196 mAfUmUmGmAfGmAmGmAmAmGm CmUmUmCmUmCmUmCmAmAmUUmCfCmAfCmCmAmCmGmAmGmU 149 mUmGfCmAmCmUfUfCfGmCm 197mAfGmAmGmGmUmGmAmAmGmCm UfUmCmAmCmCfUmCmU GmAfAmGmUmGmCmAmCmA 150mUmGfCmCmGmAfUfCfCmAm 198 mUfCmCmGmCmAmGmUmAmUmGm UfAmCmUmGmCfGmGmAGmAfUmCmGmGmCmAmGmA 151 mUmGfCmCmGmAfUfCfCmAm 199mUfCmCmGmCmAmGmUmAmUmGm UfAmCmUmGmCfGmGmA GmAfUmCmGmGmCmA 152mUmGfCmUmAmUfGfCfCmUm 200 mAfAmGmAmAmGmAmUmGmAmGm CfAmUmCmUmUfCmUmUGmCfAmUmAmGmCmAmGmC 153 mUmGfUmGmCmAfCfUfUmCm 201mAfGmGmUmGmAmAmGmCmGmAm GfCmUmUmCmAfCmCmU AmGfUmGmCmAmCmAmCmG 154mUmGfUmGmCmAfCfUmUmCm 201 mAfGmGmUmGmAmAmGmCmGmAm GmCmUmUmCmAfCmCmUAmGfUmGmCmAmCmAmCmG 155 mUmGfUmGmCmAfCfUfUmCm 202mAfGmGmUmGmAmAmGmCmGmAm GfCmUmUmCmAfCmCmU AmGfUmGmCmAmCmA 156mUmGfUmGmCmAfCfUmUmCm 202 mAfGmGmUmGmAmAmGmCmGmAm GmCmUmUmCmAfCmCmUAmGfUmGmCmAmCmA 157 mUmUfGmCmCmCfGfUmUmUm 203 mUfUmAmGmAmGmGmAmCmAmAmGmUmCmCmUmCfUmAmA AmCfGmGmGmCmAmAmCmA 158 mUmUfGmCmCmCfGfUfUmUm 204mUfUmAmGmAmGmGmAmCmAmAm GfUmCmCmUmCfUmAmA AmCfGmGmGmCmAmA mX= 2′-O-methyl nucleotide; fX = 2′-fluoro nucleotide

TABLE 3 2′-O-methyl and 2′-fluoro Modified NucleotideSequences with Phosphorothioate Linkages First NucleotideSecond Nucleotide SEQ ID NO. Sequence (5′-3′) SEQ ID NO. Sequence (5′-3′205 mApsmCpsfCmGmUmGfUfGfC 261 mGpsfApsmAmGmCmGmAmAmGmmAmCfUmUmCmGmCfUmUmC UmGmCmAfCmAmCmGmGmUpsmC psmC 206mApsmCpsfCmGmUmGfUfGfC 262 mGpsfApsmAmGmCmGmAmAmGm mAmCfUmUmCmGmCfUmUmCUmGmCmAfCmAmCmGmGmU 207 mApsmCpsfUmUmCmGfCfUfU 263mUpsfGpsmCmAmGmAmGmGmUm mCmAfCmCmUmCmUfGmCmA GmAmAmGfCmGmAmAmGmUpsmGpsmC 208 mApsmGpsfUmGmUmUfUfGfC 264 mGpsfGpsmUmUmGmCmGmUmCmmUmGfAmCmGmCmAfAmCmC AmGmCmAfAmAmCmAmCmUpsmU psmG 209mCpsmApsfGmGmCmGfGfGfG 265 mApsfCpsmAmAmGmAmAmAmAm mUmUfUmUmUmCmUfUmGmUAmCmCmCfCmGmCmCmUmGpsmU psmA 210 mCpsmApsfGmGmCmGfGfGfG 266mApsfCpsmAmAmGmAmAmAmAm mUmUfUmUmUmCmUfUmGmU AmAmCmCmCfCmGmCmCmUmG 211mCpsmApsfGmUmUmUfAfCfU 267 mApsfApsmAmUmGmGmCmAmCm mAmGfUmGmCmCmAfUmUmUUmAmGmUfAmAmAmCmUmGpsmA psmG 212 mCpsmApsfGmUmUmUfAfCfU 268mApsfApsmAmUmGmGmCmAmCm mAmGfUmGmCmCmAfUmUmU UmAmGmUfAmAmAmCmUmG 213mCpsmApsfUmCmCmUfGfCfU 269 mGpsfApsmGmGmCmAmUmAmGm mGmCfUmAmUmGmCfCmUmCCmAmGmCfAmGmGmAmUmGpsmA psmA 214 mCpsmApsmUmCmCmUfGmCf 270mApsfUpsmGmAmGfGmCmAmUm UfGfCmUmAmUmGmCmCmUmCm AmGmCmAfGmCfAmGmGmAmUmGAmU psmApsmA 215 mCpsmApsfUmCmCmUfGfCfU 271 mGpsfApsmGmGmCmAmUmAmGmmGmCfUmAmUmGmCfCmUmC CmAmGmCfAmGmGmAmUmG 216 mCpsmCpsfGmUmGmUfGfCfA 272mUpsfGpsmAmAmGmCmGmAmAm mCmUfUmCmGmCmUfUmCmA GmUmGmCfAmCmAmCmGmGpsmUpsmC 217 mCpsmCpsfGmUmGmUfGfCfA 273 mUpsfGpsmAmAmGmCmGmAmAmmCmUfUmCmGmCmUfUmCmA GmUmGmCfAmCmAmCmGmG 218 mCpsmCpsmUmGmCmUfGmCf 274mApsfApsmGmAmUfGmAmGmGm UfAfUmGmCmCmUmCmAmUmCm CmAmUmAfGmCfAmGmCmAmGmGUmU psmApsmU 219 mCpsmUpsfCmAmGmUfUfUfA 275 mApsfUpsmGmGmCmAmCmUmAmmCmUfAmGmUmGmCfCmAmU GmUmAmAfAmCmUmGmAmGpsmC psmC 220mCpsmUpsmCmAmGmUfUmU 275 mApsfUpsmGmGmCmAmCmUmAm mAmCmUmAmGmUmGmCmCmAmUGmUmAmAfAmCmUmGmAmGpsmC psmC 221 mCpsmUpsfCmAmGmUfUfUmA 275mApsfUpsmGmGmCmAmCmUmAm mCmUmAmGmUmGmCfCmAmU GmUmAmAfAmCmUmGmAmGpsmCpsmC 222 mCpsmUpsfCmAmGmUfUfUfA 276 mApsfUpsmGmGmCmAmCmUmAmmCmUfAmGmUmGmCfCmAmU GmUmAmAfAmCmUmGmAmG 223 mCpsmUpsfCmAmGmUfUfUmA 276mApsfUpsmGmGmCmAmCmUmAm mCmUmAmGmUmGmCfCmAmU GmUmAmAfAmCmUmGmAmG 224mCpsmUpsfGmCmUmAfUfGfC 277 mApsfGpsmAmAmGmAmUmGmAm mCmUfCmAmUmCmUfUmCmUGmGmCmAfUmAmGmCmAmGpsmC psmA 225 mCpsmUpsfGmCmUmAfUfGmC 277mApsfGpsmAmAmGmAmUmGmAm mCmUmCmAmUmCmUfUmCmU GmGmCmAfUmAmGmCmAmGpsmCpsmA 226 mCpsmUpsfGmCmUmAfUfGfC 278 mApsfGpsmAmAmGmAmUmGmAmmCmUfCmAmUmCmUfUmCmU GmGmCmAfUmAmGmCmAmG 227 mCpsmUpsfGmCmUmAfUfGmC 278mApsfGpsmAmAmGmAmUmGmAm mCmUmCmAmUmCmUfUmCmU GmGmCmAfUmAmGmCmAmG 228mCpsmUpsfGmCmUmGfCfUfA 279 mApsfGpsmAmUmGmAmGmGmCm mUmGfCmCmUmCmAfUmCmUAmUmAmGfCmAmGmCmAmGpsmG psmA 229 mCpsmUpsfGmCmUmGfCfUfA 280mApsfGpsmAmUmGmAmGmGmCm mUmGfCmCmUmCmAfUmCmU AmUmAmGfCmAmGmCmAmG 230mCpsmUpsfGmCmUmGfCfUmA 280 mApsfGpsmAmUmGmAmGmGmCm mUmGmCmCmUmCmAfUmCmUAmUmAmGfCmAmGmCmAmG 231 mCpsmUpsmUmCmGmCfUmUf 281mApsfCpsmGmUmGfCmAmGmAm CfAfCmCmUmCmUmGmCmAmCm GmGmUmGfAmAfGmCmGmAmAmGGmU psmUpsmG 232 mGpsmCpsmAmCmUmUfCmGf 282 mUpsfGpsmCmAmGfAmGmGmUmCfUfUmCmAmCmCmUmCmUmGm GmAmAmGfCmGfAmAmGmUmGmC CmA psmApsmC 233mGpsmCpsfCmGmAmUfCfCfA 283 mUpsfUpsmCmCmGmCmAmGmUm mUmAfCmUmGmCmGfGmAmAAmUmGmGfAmUmCmGmGmCpsmA psmG 234 mGpsmCpsfCmGmGmGfUfUfU 284mUpsfUpsmCmCmGmCmAmGmUm mUmUfCmUmUmGmUfUmGmA AmUmGmGfAmUmCmGmGmC 235mGpsmCpsfGmGmGmGfUfUfU 285 mUpsfCpsmAmAmCmAmAmGmAm mUmUfCmUmUmGmUfUmGmAAmAmAmAfCmCmCmCmGmCpsmC psmU 236 mGpsmCpsfGmGmGmGfUfUmU 285mUpsfCpsmAmAmCmAmAmGmAm mUmUmCmUmUmGmUfUmGmA AmAmAmAfCmCmCmCmGmCpsmCpsmU 237 mGpsmCpsfGmGmGmGfUfUfU 286 mUpsfCpsmAmAmCmAmAmGmAmmUmUfCmUmUmGmUfUmGmA AmAmAmAfCmCmCmCmGmC 238 mGpsmCpsfGmGmGmGfUfUmU 286mUpsfCmAmAmCmAmAmGmAmA mUmUmCmUmUmGmUfUmGmA mAmAmAfCmCmCmCmGmC 239mGpsmCpsmUmGmCmUfAmUf 287 mApsfApsmGmAmAfGmAmUmGm GfCfCmUmCmAmUmCmUmUmCmAmGmGmCfAmUfAmGmCmAmGmC UmU psmApsmG 240 mGpsmGpsmAmUmGmUfGmUf 288mUpsfApsmAmAmAfCmGmCmCm CfUfGmCmGmGmCmGmUmUmUm GmCmAmGfAmCfAmCmAmUmCmCUmA psmApsmG 241 mGpsmGpsfCmCmAmAfAfAfU 289 mGpsfGpsmGmAmCmUmGmCmGmmUmCfGmCmAmGmUfCmCmC AmAmUmUfUmUmGmGmCmCpsmA psmA 242mGpsmGpsfCmGmCmAfCfCfU 290 mGpsfCpsmGmUmAmAmAmGmAm mCmUfCmUmUmUmAfCmGmCGmAmGmGfUmGmCmGmCmCpsmC psmC 243 mGpsmUpsfAmUmGmUfUfGfC 291mGpsfGpsmAmCmAmAmAmCmGm mCmCfGmUmUmUmGfUmCmC GmGmCmAfAmCmAmUmAmCpsmCpsmU 244 mGpsmUpsfGmGmUmGfGfAfC 292 mApsfUpsmUmGmAmGmAmGmAmmUmUfCmUmCmUmCfAmAmU AmGmUmCfCmAmCmCmAmCpsmG psmA 245mGpsmUpsfGmUmGmCfAfCfU 293 mGpsfGpsmUmGmAmAmGmCmGm mUmCfGmCmUmUmCfAmCmCAmAmGmUfGmCmAmCmAmCpsmG psmG 246 mGpsmUpsfUmGmCmCfCfGfU 294mUpsfApsmGmAmGmGmAmCmAm mUmUfGmUmCmCmUfCmUmA AmAmCmGfGmGmCmAmAmCpsmApsmU 247 mGpsmUpsfUmGmCmCfCfGfU 295 mUpsfApsmGmAmGmGmAmCmAmmUmUfGmUmCmCmUfCmUmA AmAmCmGfGmGmCmAmAmC 248 mUpsmCpsfCmAmUmAfCfUfG 296mApsfGpsmGmAmGmUmUmCmCm mCmGfGmAmAmCmUfCmCmU GmCmAmGfUmAmUmGmGmApsmUpsmC 249 mUpsmCpsfCmAmUmAfCfUfG 297 mApsfGpsmGmAmGmUmUmCmCmmCmGfGmAmAmCmUfCmCmU GmCmAmGfUmAmUmGmGmA 250 mUpsmCpsmGmUmGmGfUmGf 298mApsfUpsmUmGmAfGmAmGmAm GfAfCmUmUmCmUmCmUmCmAm AmGmUmCfCmAfCmCmAmCmGmAAmU psmGpsmU 251 mUpsmGpsfCmAmCmUfUfCfG 299 mApsfGpsmAmGmGmUmGmAmAmmCmUfUmCmAmCmCfUmCmU GmCmGmAfAmGmUmGmCmApsmC psmA 252mUpsmGpsfCmCmGmAfUfCfC 300 mUpsfCpsmCmGmCmAmGmUmAm mAmUfAmCmUmGmCfGmGmAUmGmGmAfUmCmGmGmCmApsmG psmA 253 mUpsmGpsfCmCmGmAfUfCfC 301mUpsfCpsmCmGmCmAmGmUmAm mAmUfAmCmUmGmCfGmGmA UmGmGmAfUmCmGmGmCmA 254mUpsmGpsfCmUmAmUfGfCfC 302 mApsfApsmGmAmAmGmAmUmGm mUmCfAmUmCmUmUfCmUmUAmGmGmCfAmUmAmGmCmApsmG psmC 255 mUpsmGpsfUmGmCmAfCfUfU 303mApsfGpsmGmUmGmAmAmGmCm mCmGfCmUmUmCmAfCmCmU GmAmAmGfUmGmCmAmCmApsmCpsmG 256 mUpsmGpsfUmGmCmAfCfUmU 303 mApsfGpsmGmUmGmAmAmGmCmmCmGmCmUmUmCmAfCmCmU GmAmAmGfUmGmCmAmCmApsmC psmG 257mUpsmGpsfUmGmCmAfCfUfU 304 mApsfGpsmGmUmGmAmAmGmCm mCmGfCmUmUmCmAfCmCmUGmAmAmGfUmGmCmAmCmA 258 mUpsmGpsfUmGmCmAfCfUmU 304mApsfGpsmGmUmGmAmAmGmCm mCmGmCmUmUmCmAfCmCmU GmAmAmGfUmGmCmAmCmA 259mUpsmUpsfGmCmCmCfGfUmU 305 mUpsfUpsmAmGmAmGmGmAmCm mUmGmUmCmCmUmCfUmAmAAmAmAmCfGmGmGmCmAmApsmC psmA 260 mUpsmUpsfGmCmCmCfGfUfU 306mUpsfUpsmAmGmAmGmGmAmCm mUmGfUmCmCmUmCfUmAmA AmAmAmCfGmGmGmCmAmA mX= 2′-O-methyl nucleotide; fX = 2′-fluoro nucleotide; ps= phosphorothioate linkage

TABLE 4 siNA Sequences SEQ ID SEQ NO. Sense Sequence (5′-3′) ID NO.Antisense Sequence (5′-3′) 307 mApsmCpsfCmGmUmGfUfGfC 363mGpsfApsmAmGmCmGmAmAmGmU mAmCfUmUmCmGmCfUmUmC mGmCmAfCmAmCmGmGmUpsmCpsmC 308 mApsmCpsfCmGmUmGfUfGfC 364 mGpsfApsmAmGmCmGmAmAmGmUmAmCfUmUmCmGmCfUmUmC mGmCmAfCmAmCmGmGmUpsTpsT TT 309mApsmCpsfUmUmCmGfCfUfU 365 mUpsfGpsmCmAmGmAmGmGmUmG mCmAfCmCmUmCmUfGmCmAmAmAmGfCmGmAmAmGmUpsmGps mC 310 mApsmGpsfUmGmUmUfUfGfC 366mGpsfGpsmUmUmGmCmGmUmCmA mUmGfAmCmGmCmAfAmCmC mGmCmAfAmAmCmAmCmUpsmUpsmG 311 mCpsmApsfGmGmCmGfGfGfG 367 mApsfCpsmAmAmGmAmAmAmAmAmUmUfUmUmUmCmUfUmGm mCmCmCfCmGmCmCmUmGpsmUps U mA 312mCpsmApsfGmGmCmGfGfGfG 368 mApsfCpsmAmAmGmAmAmAmAmA mUmUfUmUmUmCmUfUmGmmAmCmCmCfCmGmCmCmUmGpsTp UTT sT 313 mCpsmApsfGmUmUmUfAfCfU 369mApsfApsmAmUmGmGmCmAmCmU mAmGfUmGmCmCmAfUmUm mAmGmUfAmAmAmCmUmGpsmAps UmG 314 mCpsmApsfGmUmUmUfAfCfU 370 mApsfApsmAmUmGmGmCmAmCmUmAmGfUmGmCmCmAfUmUm mAmGmUfAmAmAmCmUmGpsTpsT UTT 315mCpsmApsfUmCmCmUfGfCfU 371 mGpsfApsmGmGmCmAmUmAmGmC mGmCfUmAmUmGmCfCmUmCmAmGmCfAmGmGmAmUmGpsmAps mA 316 mCpsmApsmUmCmCmUfGmCf 372mApsfUpsmGmAmGfGmCmAmUmA UfGfCmUmAmUmGmCmCmUm mGmCmAfGmCfAmGmGmAmUmGpsCmAmU mApsmA 317 mCpsmApsfUmCmCmUfGfCfU 373 mGpsfApsmGmGmCmAmUmAmGmCmGmCfUmAmUmGmCfCmUmC mAmGmCfAmGmGmAmUmGpsTpsT TT 318mCpsmCpsfGmUmGmUfGfCfA 374 mUpsfGpsmAmAmGmCmGmAmAmG mCmUfUmCmGmCmUfUmCmAmUmGmCfAmCmAmCmGmGpsmUps mC 319 mCpsmCpsfGmUmGmUfGfCfA 375mUpsfGpsmAmAmGmCmGmAmAmG mCmUfUmCmGmCmUfUmCmA mUmGmCfAmCmAmCmGmGpsTpsTTT 320 mCpsmCpsmUmGmCmUfGmCf 376 mApsfApsmGmAmUfGmAmGmGmCUfAfUmGmCmCmUmCmAmUm mAmUmAfGmCfAmGmCmAmGmGps CmUmU mApsmU 321mCpsmUpsfCmAmGmUfUfUfA 377 mApsfUpsmGmGmCmAmCmUmAmG mCmUfAmGmUmGmCfCmAmUmUmAmAfAmCmUmGmAmGpsmCps mC 322 mCpsmUpsmCmAmGmUfUmU 377mApsfUpsmGmGmCmAmCmUmAmG mAmCmUmAmGmUmGmCmC mUmAmAfAmCmUmGmAmGpsmCpsmAmU mC 323 mCpsmUpsfCmAmGmUfUfUmA 377 mApsfUpsmGmGmCmAmCmUmAmGmCmUmAmGmUmGmCfCmAm mUmAmAfAmCmUmGmAmGpsmCps U mC 324mCpsmUpsfCmAmGmUfUfUfA 378 mApsfUpsmGmGmCmAmCmUmAmG mCmUfAmGmUmGmCfCmAmUmUmAmAfAmCmUmGmAmGpsTpsT TT 325 mCpsmUpsfCmAmGmUfUfUmA 378mApsfUpsmGmGmCmAmCmUmAmG mCmUmAmGmUmGmCfCmAm mUmAmAfAmCmUmGmAmGpsTpsTUTT 326 mCpsmUpsfGmCmUmAfUfGfC 379 mApsfGpsmAmAmGmAmUmGmAmGmCmUfCmAmUmCmUfUmCmU mGmCmAfUmAmGmCmAmGpsmCps mA 327mCpsmUpsfGmCmUmAfUfGmC 379 mApsfGpsmAmAmGmAmUmGmAmG mCmUmCmAmUmCmUfUmCmmGmCmAfUmAmGmCmAmGpsmCps U mA 328 mCpsmUpsfGmCmUmAfUfGfC 380mApsfGpsmAmAmGmAmUmGmAmG mCmUfCmAmUmCmUfUmCmU mGmCmAfUmAmGmCmAmGpsTpsTTT 329 mCpsmUpsfGmCmUmAfUfGmC 380 mApsfGpsmAmAmGmAmUmGmAmGmCmUmCmAmUmCmUfUmCm mGmCmAfUmAmGmCmAmGpsTpsT UTT 330mCpsmUpsfGmCmUmGfCfUfA 381 mApsfGpsmAmUmGmAmGmGmCmA mUmGfCmCmUmCmAfUmCmUmUmAmGfCmAmGmCmAmGpsmGps mA 331 mCpsmUpsfGmCmUmGfCfUfA 382mApsfGpsmAmUmGmAmGmGmCmA mUmGfCmCmUmCmAfUmCmU mUmAmGfCmAmGmCmAmGpsTpsTTT 332 mCpsmUpsfGmCmUmGfCfUmA 383 mApsfGpsmAmUmGmAmGmGmCmAmUmGmCmCmUmCmAfUmCm mUmAmGfCmAmGmCmAmGpsTpsT UTT 333mCpsmUpsmUmCmGmCfUmUf 384 mApsfCpsmGmUmGfCmAmGmAmG CfAfCmCmUmCmUmGmCmAmmGmUmGfAmAfGmCmGmAmAmGp CmGmU smUpsmG 334 mGpsmCpsmAmCmUmUfCmGf 385mUpsfGpsmCmAmGfAmGmGmUmG CfUfUmCmAmCmCmUmCmUm mAmAmGfCmGfAmAmGmUmGmCpsGmCmA mApsmC 335 mGpsmCpsfCmGmAmUfCfCfA 386 mUpsfUpsmCmCmGmCmAmGmUmAmUmAfCmUmGmCmGfGmAm mUmGmGfAmUmCmGmGmCpsmAps A mG 336mGpsmCpsfCmGmGmGfUfUfU 387 mUpsfUpsmCmCmGmCmAmGmUmA mUmUfC mUmUmGmUfUmGmmUmGmGfAmUmCmGmGmCpsTpsT ATT 337 mGpsmCpsfGmGmGmGfUfUfU 388mUpsfCpsmAmAmCmAmAmGmAmA mUmUfC mUmUmGmUfUmGm mAmAmAfCmCmCmCmGmCpsmCps AmU 338 mGpsmCpsfGmGmGmGfUfUmU 388 mUpsfCpsmAmAmCmAmAmGmAmAmUmUmCmUmUmGmUfUmGm mAmAmAfCmCmCmCmGmCpsmCps A mU 339mGpsmCpsfGmGmGmGfUfUfU 389 mUpsfCpsmAmAmCmAmAmGmAmA mUmUfCmUmUmGmUfUmGmmAmAmAfCmCmCmCmGmCpsTpsT ATT 340 mGpsmCpsfGmGmGmGfUfUmU 389mUpsfCmAmAmCmAmAmGmAmAm mUmUmCmUmUmGmUfUmGm AmAmAfCmCmCmCmGmCpsTpsT ATT341 mGpsmCpsmUmGmCmUfAmUf 390 mApsfApsmGmAmAfGmAmUmGmAGfCfCmUmCmAmUmCmUmUm mGmGmCfAmUfAmGmCmAmGmCps CmUmU mApsmG 342mGpsmGpsmAmUmGmUfGmUf 391 mUpsfApsmAmAmAfCmGmCmCmG CfUfGmCmGmGmCmGmUmUmmCmAmGfAmCfAmCmAmUmCmCps UmUmA mApsmG 343 mGpsmGpsfCmCmAmAfAfAfU 392mGpsfGpsmGmAmCmUmGmCmGmA mUmCfGmCmAmGmUfCmCmC mAmUmUfUmUmGmGmCmCpsmApsmA 344 mGpsmGpsfCmGmCmAfCfCfU 393 mGpsfCpsmGmUmAmAmAmGmAmGmCmUfCmUmUmUmAfCmGmC mAmGmGfUmGmCmGmCmCpsmCps mC 345mGpsmUpsfAmUmGmUfUfGfC 394 mGpsfGpsmAmCmAmAmAmCmGmG mCmCfGmUmUmUmGfUmCmCmGmCmAfAmCmAmUmAmCpsmCps mU 346 mGpsmUpsfGmGmUmGfGfAfC 395mApsfUpsmUmGmAmGmAmGmAmA mUmUfCmUmCmUmCfAmAmU mGmUmCfCmAmCmCmAmCpsmGpsmA 347 mGpsmUpsfGmUmGmCfAfCfU 396 mGpsfGpsmUmGmAmAmGmCmGmAmUmCfGmCmUmUmCfAmCmC mAmGmUfGmCmAmCmAmCpsmGps mG 348mGpsmUpsfUmGmCmCfCfGfU 397 mUpsfApsmGmAmGmGmAmCmAmA mUmUfGmUmCmCmUfCmUmAmAmCmGfGmGmCmAmAmCpsmAps mU 349 mGpsmUpsfUmGmCmCfCfGfU 398mUpsfApsmGmAmGmGmAmCmAmA mUmUfGmUmCmCmUfCmUmA mAmCmGfGmGmCmAmAmCpsTpsTTT 350 mUpsmCpsfCmAmUmAfCfUfG 399 mApsfGpsmGmAmGmUmUmCmCmGmCmGfGmAmAmCmUfCmCmU mCmAmGfUmAmUmGmGmApsmUps mC 351mUpsmCpsfCmAmUmAfCfUfG 400 mApsfGpsmGmAmGmUmUmCmCmG mCmGfGmAmAmCmUfCmCmUmCmAmGfUmAmUmGmGmApsTpsT TT 352 mUpsmCpsmGmUmGmGfUmGf 401mApsfUpsmUmGmAfGmAmGmAmA GfAfCmUmUmCmUmCmUmCm mGmUmCfCmAfCmCmAmCmGmApsAmAmU mGpsmU 353 mUpsmGpsfCmAmCmUfUfCfG 402 mApsfGpsmAmGmGmUmGmAmAmGmCmUfUmCmAmCmCfUmCmU mCmGmAfAmGmUmGmCmApsmCps mA 354mUpsmGpsfCmCmGmAfUfCfC 403 mUpsfCpsmCmGmCmAmGmUmAmU mAmUfAmCmUmGmCfGmGmmGmGmAfUmCmGmGmCmApsmGps A mA 355 mUpsmGpsfCmCmGmAfUfCfC 404mUpsfCpsmCmGmCmAmGmUmAmU mAmUfAmCmUmGmCfGmGm mGmGmAfUmCmGmGmCmApsTpsTATT 356 mUpsmGpsfCmUmAmUfGfCfC 405 mApsfApsmGmAmAmGmAmUmGmAmUmCfAmUmCmUmUfCmUmU mGmGmCfAmUmAmGmCmApsmGps mC 357mUpsmGpsfUmGmCmAfCfUfU 406 mApsfGpsmGmUmGmAmAmGmCmG mCmGfCmUmUmCmAfCmCmUmAmAmGfUmGmCmAmCmApsmCps mG 358 mUpsmGpsfUmGmCmAfCfUmU 406mApsfGpsmGmUmGmAmAmGmCmG mCmGmCmUmUmCmAfCmCm mAmAmGfUmGmCmAmCmApsmCps UmG 359 mUpsmGpsfUmGmCmAfCfUfU 407 mApsfGpsmGmUmGmAmAmGmCmGmCmGfCmUmUmCmAfCmCmU mAmAmGfUmGmCmAmCmApsTpsT TT 360mUpsmGpsfUmGmCmAfCfUmU 407 mApsfGpsmGmUmGmAmAmGmCmG mCmGmCmUmUmCmAfCmCmmAmAmGfUmGmCmAmCmApsTpsT UTT 361 mUpsmUpsfGmCmCmCfGfUmU 408mUpsfUpsmAmGmAmGmGmAmCmA mUmGmUmCmCmUmCfUmAm mAmAmCfGmGmGmCmAmApsmCps AmA 362 mUpsmUpsfGmCmCmCfGfUfU 409 mUpsfUpsmAmGmAmGmGmAmCmAmUmGfUmCmCmUmCfUmAmA mAmAmCfGmGmGmCmAmApsTpsT TT 4155dcd3Cps5dcd3CpsfG5dcd3UmG 445 5dcd3UpsfGpsmAmAmG5dcd3CmGm5dcd3UfG5dfCfA5dcd3C5dcd3U AmAmG5dcd3UmG5dcd3CfA5dcd3C5dfU5dcd3CmG5dcd3C5dcd3U5d mA5dcd3CmGmGps5dcd3Ups5dcd3U fU5dcd3CmA 4155dcd3Cps5dcd3CpsfG5dcd3UmG 446 5dcd3UpsfGpsmAmAmGmCmGmAm5dcd3UfG5dfCfA5dcd3C5dcd3U AmGmUmGmCfAmCmAmCmGmGps5dfU5dcd3CmG5dcd3C5dcd3U5d 5dcd3Ups5dcd3U fU5dcd3CmA 4155dcd3Cps5dcd3CpsfG5dcd3UmG 447 mUpsfGpsmAmAmGmCmGmAmAmG5dcd3UfG5dfCfA5dcd3C5dcd3U mUmGmCfAmCmAmCmGmGpsmUps5dfU5dcd3CmG5dcd3C5dcd3U5d mU fU5dcd3CmA 416 fCpsmCpsfGmUfGmUfGfCfAmC448 mUpsfGpsmAfAmGfCmGfAmAfGmU fUmUfCmGfCmUfUmCfAmGmCfAmCfAmCfGmGpsmUpsmC 416 fCpsmCpsfGmUfGmUfGfCfAmC 449vmUpsfGpsmAfAmGfCmGfAmAfGm fUmUfCmGfCmUfUmCfA UmGmCfAmCfAmCfGmGpsmUpsmC416 fCpsmCpsfGmUfGmUfGmCfAm 450 mUpsfGpsmAfAmGfCmGfAmAfGmUfCfUmUfCmGfCmUfUmCfA GmCfAmCfAmCfGmGpsmUpsmC 416 fCpsmCpsfGmUfGmUfGmCfAm451 vmUpsfGpsmAfAmGfCmGfAmAfGm CfUmUfCmGfCmUfUmCfAUfGmCfAmCfAmCfGmGpsmUpsmC 417 fCpsmUpsfGmCfUmAfUmGfCm 452mApsfGpsmAfAmGfAmUfGmAfGmGf CfUmCfAmUfCmUfUmCfU CmAfUmAfGmCfAmGpsmUpsmU417 fCpsmUpsfGmCfUmAfUmGfCm 452 mApsfGpsmAfAmGfAmUfGmAfGmGfCfUmCfAmUfCmUfUmCfU CmAfUmAfGmCfAmGpsmUpsmU 417 fCpsmUpsfGmCfUmAfUmGfCm452 mApsfGpsmAfAmGfAmUfGmAfGmGf CfUmCfAmUfCmUfUmCfUCmAfUmAfGmCfAmGpsmUpsmU 417 fCpsmUpsfGmCfUmAfUmGfCm 452mApsfGpsmAfAmGfAmUfGmAfGmGf CfUmCfAmUfCmUfUmCfU CmAfUmAfGmCfAmGpsmUpsmU418 fGpsmUpsfGmGfUmGfGfAfCm 453 mApsfUpsmUfGmAfGmAfGmAfAmGUfUmCfUmCfUmCfAmAfU mUmCfCmAfCmCfAmCpsmGpsmA 418 fGpsmUpsfGmGfUmGfGfAfCm454 vmApsfUpsmUfGmAfGmAfGmAfAm UfUmCfUmCfUmCfAmAfUGmUmCfCmAfCmCfAmCpsmGpsmA 419 fGpsmUpsfGmGfUmGfGmAfCm 455mApsfUpsmUfGmAfGmAfGmAfAmGf UfUmCfUmCfUmCfAmAfU UmCfCmAfCmCfAmCpsmGpsmA419 fGpsmUpsfGmGfUmGfGmAfCm 456 vmApsfUpsmUfGmAfGmAfGmAfAmUfUmCfUmCfUmCfAmAfU GfUmCfCmAfCmCfAmCpsmGpsmA 420mCpsmCpsfGmUfGmUfGfCfAm 457 mUpsfGpsmAmAmGfCmGmAmAmG CmUmUmCmGmCmUmUmCmmUmGmCfAmCfAmCmGmGpsmUps A mC 421 mCpsmCpsfGmUmGmUfGfCfA 458mUpsfGpsmAmAmGmCmGmAmAmG mAmUfUmCmGmCmUfUmCmA mUmGmCfAmCmAmCmGmGpsmUpsmC 422 mCpsmCpsfGmUmGmUfGfCfA 459 mUpsfGpsmAmAmGmCmGmAmAmGmCmUfUmCmGmCmUfUmCmA mUmGmCfAmCmAmCmGmGpsmU3s 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533 mApsfGpsmGmUmGmAmAmGmCmG mCmGmCmUmUmCmAfCmCmmAmAmGfUmGmCmAmCmApsTpsT U 424 mCpsmCpsmGmUfGmUfGfCfA 536d2vd3UpsfGpsmAmAfGmCmGfAmAm mCmUmUmCmGmCmUmUmC GmUmGmCfAmCmAfCmGmGpsmUpmA smC 438 mGpsmUpsmGmGfUmGfGfAfC 537 mApsf4PpsmUmGmAfGmAmGmAmAmUmUmCmUmCmUmCmAmA mGmUmCfCmAfCmCmAmCpsmGpsm mU A 438mGpsmUpsmGmGfUmGfGfAfC 538 mApsfUpsmUmGmAfGmAmGmAmA mUmUmCmUmCmUmCmAmAmGmUmCf2PmAfCmCmAmCpsmGps mU mA 438 mGpsmUpsmGmGfUmGfGfAfC 599mApsfUpsmUmGmAfGmAmGmAmA mUmUmCmUmCmUmCmAmA mGmUmCfCmAfXmCmAmCpsmGps mUmA mX = 2′-O-methyl nucleotide; fX = 2′-fluoro nucleotide; 5dcd3X =nucleotide of Formula 17; 5dfX = nucleotide of Formula 16; vX = 5′ vinylphosphonate nucleotide; d2vX = deuterated 5′ vinyl phosphonatenucleotide; vmX = 5′ vinyl phosphonate, 2′-O-methylnucleotide; vmB =

vmN =

VmU =

cmU =

mesnmU =

mesnomU =

d2vmU =

d2vmA =

d2vd3U =

f4P =

f2P =

fX =

ps = phosphorothioate linkage

TABLE 5 SEQ ID NO: Description Sequence⁺ 410 Hepatitis Baattccacaacctttcaccaaactctgcaagatcccagagtgagaggcctgtatttccctgctggtggvirusctccagttcaggagcagtaaaccctgttccgactactgcctctcccttatcgtcaatcttctcgaggatt(GenbankggggaccctgcgctgaacatggagaacatcacatcaggattcctaggaccccttctcgtgttacaggAccessioncggggtttttcttgttgacaagaatcctcacaataccgcagagtctagactcgtggtggacttctctcaNo. attttctagggggaactaccgtgtgtcttggccaaaattcgcagtccccaacctccaatcactcaccaU95551.1)acctcctgtcctccaacttgtcctggttatcgctggatgtgtctgcggcgttttatcatcttcctcttcatcctgctgctatgcctcatcttcttgttggttcttctggactatcaaggtatgttgcccgtttgtcctctaattccaggatcctcaaccaccagcacgggaccatgccgaacctgcatgactactgctcaaggaacctctatgtatccctcctgttgctgtaccaaaccttcggacggaaattgcacctgtattcccatcccatcatcctgggctttcggaaaattcctatgggagtgggcctcagcccgtttctcctggctcagtttactagtgccatttgttcagtggttcgtagggctttcccccactgtttggctttcagttatatggatgatgtggtattgggggccaagtctgtacagcatcttgagtccctttttaccgctgttaccaattttcttttgtctttgggtatacatttaaaccctaacaaaacaaagagatggggttactctctgaattttatgggttatgtcattggaagttatgggtccttgccacaagaacacatcatacaaaaaatcaaagaatgttttagaaaacttcctattaacaggcctattgattggaaagtatgtcaacgaattgtgggtcttttgggttttgctgccccatttacacaatgtggttatcctgcgttaatgcccttgtatgcatgtattcaatctaagcaggctttcactttctcgccaacttacaaggcctttctgtgtaaacaatacctgaacctttaccccgttgcccggcaacggccaggtctgtgccaagtgtttgctgacgcaacccccactggctggggcttggtcatgggccatcagcgcgtgcgtggaaccttttcggctcctctgccgatccatactgcggaactcctagccgcttgttttgctcgcagcaggtctggagcaaacattatcgggactgataactctgttgtcctctcccgcaaatatacatcgtatccatggctgctaggctgtgctgccaactggatcctgcgcgggacgtcctttgtttacgtcccgtcggcgctgaatcctgcggacgaccatcteggggtcgcttgggactctctcgtccccttctccgtctgccgttccgaccgaccacggggcgcacctctattacgcggactccccgtctgtgccttctcatctgccggaccgtgtgcacttcgcttcacctctgcacgtcgcatggagaccaccgtgaacgcccaccgaatgttgcccaaggtcttacataagaggactatggactctctgcaatgtcaacgaccgaccttgaggcatacttcaaagactgtttgtttaaagactgggaggagttgggggaggagattagattaaaggtetttgtactaggaggctgtaggcataaattggtctgcgcaccagcaccatgcaactttttcacctctgcctaatcatctcttgttcatgtcctactgttcaagcctccaagctgtgccttgggtggctttggggcatggacatcgacccttataaagaatttggagctactgtggagttactctcgttfttgccttctgacttctttccttcagtacgagatcttctagataccgcctcagctctgtatcgggaagccttagagtctcctgagcattgttcacctcaccatactgcactcaggcaagcaattctttgctggggggaactaatgactctagctacctgggtgggtgttaatttggaagatccagcatctagagacctagtagtcagttatgtcaacactaatatgggcctaaagttcaggcaactcttgtggtttcacatttcttgtctcacttttggaagagaaaccgttatagagtatttggtgtctttcggagtgtggattcgcactcctccagatatagaccaccaaatgcccctatcctatcaacacttccggaaactactgttgttagacgacgaggcaggtcccctagaagaagaactccctcgcctcgcagacgaaggtctcaatcgccgcgtcgcagaagatctcaatctcgggaacctcaatgttagtattccttggactcataaggtggggaactttactggtctttattcttctactgtacctgtctttaatcctcattggaaaacaccatcttttcctaatatacatttacaccaagacattatcaaaaaatgtgaacagtttgtaggcccacttacagttaatgagaaaagaagattgcaattgattatgcctgctaggttttatccaaaggttaccaaatatttaccattggataagggtattaaaccttattatccagaacatctagttaatcattacttccaaactagacactatttacacactctatggaaggcgggtatattatataagagagaaacaacacatagcgcctcattttgtgggtcaccatattcttgggaacaagatctacagcatggggcagaatctttccaccagcaatcctctgggattctttcccgaccaccagttggatccagccttcagagcaaacacagcaaatccagattgggacttcaatcccaacaaggacacctggccagacgccaacaaggtaggagctggagcattcgggctgggtttcaccccaccgcacggaggccttttggggtggagccctcaggctcagggcatactacaaactttgccagcaaatccgcctcctgcctccaccaatcgccagacaggaaggcagcctaccccgctgtctccacctttgagaaacactcatcctcaggccatgcagtgg 411 MCJ mRNAagtcactgccgcggcgccttgagtctccgggccgccttgccatggctgcccgtggtgtcatcgctc(GenBankcagttggcgagagtttgcgctacgctgagtacttgcagccctcggccaaacggccagacgccgacAccessiongtcgaccagcagagactggtaagaagtttgatagctgtaggactgggtgttgcagctcttgcatttgc No.aggtcgctacgcatttcggatctggaaacctctagaacaagttatcacagaaactgcaaagaagatttNM_013238.3)caactcctagcttttcatcctactataaaggaggatttgaacagaaaatgagtaggcgagaagctggtcttattttaggtgtaagcccatctgctggcaaggctaagattagaacagctcataggagagtcatgattttgaatcacccagataaaggtggatctccttacgtagcagccaaaataaatgaagcaaaagacttgctagaaacaaccaccaaacattgatgcttaaggaccacactgaaggaaaaaaaaagaggggacttcgaaaaaaaaaaaagccctgcaaaatattctaaaacatggtcttcttaattttctatatggattgaccacagtcttatcttccaccattaagctgtataacaataaaatgttaatagtcttgctttttattatcttttaaagatctccttaaattctataactgatcttttttcttattttgtttgtgacattcatacatttttaagatttttgttatgttctgaattcccccctacacacacacacacacacacacacacacacacgtgcaaaaaatatgatcaagaatgcaattgggatttgtgagcaatgagtagacctcttattgtttatatttgtaccctcattgtcaatttttttttagggaatttgggactctgcctatataaggtgttttaaatgtcttgagaacaagcactggctgatacctcttggagatatgatctgaaatgtaatggaatttattaaatggtgtttagtaaagtaggggttaaggacttgttaaagaaccccactatctctgagaccctatagccaaagcatgaggacttggagagctactaaaatgattcaggtttacaaaatgagccctgtgaggaaaggttgagagaagtctgaggagtttgtatttaattatagtcttccagtactgtatattcattcattactcattctacaaatatttattgaccccttttgatgtgcaaggcactatcgtgcgtcccctgagagttgcaagtatgaagcagtcatggatcatgaaccaaaggaacttatatgtagaggaaggataaatcacaaatagtgaatactgttagatacagatgatatattttaaaagttcaaaggaagaaaagaatgtgttaaacactgcatgagaggaggaataagtggcatagagctaggctttagaaaagaaaaatattccgataccatatgattggtgaggtaagtgttattctgagatgagaattagcagaaatagatatatcaatcggagtgattagagtgcagggtttctggaaagcaaggtttggacagagtggtcatcaaaggccagccctgtgacttacactgcattaaattaatttcttagaacatagtccctgatcattatcactttactattccaaaggtgagagaacagattcagatagagtgccagcattgtttcccagtattcctttacaaatcttgggttcattccaggtaaactgaactactgcattgtttctatcttaaaatactttttagatatcctagatgcatcttcaacttctaacattctgtagtttaggagttctcaaccttggcattattgacatgttaggccaaataattttttttgtgggaggtctcttgtgcgttttagatgattagcaataatccctgacctgttatctactaaagactagtcgtttctcatcagttgtgacaacaaaaatggttccagatattgccaaatgccctttagaggacagtaatcgcccccagttgagaaccatttcagtaaaactttaattactattttttcttttggtttataaaataatgatcctgaattaaattgatggaaccttgaagtcgataaaatatatttcttgctttaaagtccccatacgtgtcctactaattttctcatgctttagtgttttcacttttctcctgttatccttgtacctaagaatgccatcccaatccccagatgtccacctgcccaaagtctaggcatagctgaaggccaagctaaaatgtatccctctttttctggtacatgcagcaaaagtaatatgaattatcagctttctgagagcaggcattgtatctgtcttgtttggtgttacattggcacccaataaatatttgttgagtgaatgaataaattcccatagcactttattcttcacatggtacataactataggggctatagcttggtaccttgtgaagcaactcttggtgtaacataccttatttctcatactaaaatgcaagaacctttagagcaaggatcttgccattcatctttgtaacctctttactctggagcacttgcatttagcaggcatcataaagttttacgtaccaagaaaatgttgctgttttctgaatactatgcatcaaaaaatgttaccactaatttttaaagctctgctaaggaatattggggcaccctcagatgcaccttttaattgatgtcatattttcctaatccatactttattcatgagaatttgagtcaccccagcattagcttggaatttccttatttcccatttgctttgcaggtgccttggagtcagatctggttttgaatactatcttcctgttatgtgatcttgggcagttacttaattttctagtcaataacccgtatctataaaatagagaaaataatcctacacaccggggcctgttgtggggcggggagaggggggagggatcgcatttggagatatactaatgtaaatgacaagttaattggtgcagcacaccaacatggctcatgtctacatatgtaacaaacctgcacgttgtgcacatgtgccctagaacttaaagtataataaaaagaaattttaaaaaatcctgtcaaataaggttatagtagagaataaggatgtgtaaagcatttagtcacgtaaatgcttaaaaaaatgtaatttttacttctttcactgcctcatttaattagttttatctttaataataccttggattcagggtaaagtttcagttatgtcccagtaatcatttattttaccctcgaatctgcaatttggatagaacatggtggggacagctcgtctctattccttgcagcattaacaggctggaggcaccacttctctggccagcaagttgggcctggttgttggctgagagcctcagttcctttctgcacaggttcctctttacataggcttctcaacagggctactagagcatcgtcaccatagcagctgtcttataacagagagtggtcggtctgagagacaaaaaatggaagctgccaaattgttctgggtctggaaactgtcagggcatcacttgtgccatattcagttggcctaagaattacagagcctgcctcgattcaaagggagaggatagagaggactgaaggaatcagtgctcatctttaatatgcagcaggacaggtttgggattttttttcccccttgagtctgtgaaggcattacttaagaacaaagtcaggcatgtataattgaactacagttacttgaaatataagcccagaaagtttcagataataaatacaactatttttctgctgttacccttgtacctaaagatgccatcctaatccccagatctccacaactatacctacatagtagaaggttaaaatgtatccctctttttctggtgcatccagcaaaagtaatatcatgaattatgagctctctgagagcaaggatcatatcagtcttgtttattgttgcagtgaacaagtacagttgcagatattcaggagtaattatctaaatggcagtaggcttataaaactgaattttcaccagccacaccctccccccaactccttatctgtaaaaagcttatttgagtggttacctgtcttcagtaaagattgcgcttgcatatttgctgtcattgcatattctgcttaattaagctctgttgatattgcagtttctgtgcatacttacatcttagatgcaatctgagggcctaggaaggccttttaaaaataaaacaattccgattgcagagaaagtgtaagtcaaggacagttaattcaaggggaacatagaaagctatttagattttagttgatggtgccagtcttcagcgtaaagtcaaaagtggagggaagtttagtaaggaaaaaatgttgggcttggaatacattgtttagtcttcaaagcactttactttttatgaaatatattttagacattcagcaaatattgaatacttactatatcaggcagtaaagatataaattcattcttaaaatgtgcaacatgttcaaactgaaaaaaatacattcttaaacaggaaactttttccttcatactttttaattaacaagacatataagagttgcattaatgggcgtgcttatgattgatcacccagcagcatcattagaaataatatattttattcatgtgcagaaatcttttggttgtcctggggaaccttgaacacagaaaagagcttttattgataaggtaattgaacacacttgacaattagcttaatatggtttaataccatttgtgggagaagatgaatcagccaggctctttacgtcaagaatatgaagtttctcttgagtcaaccaacttaagatgagctacggagactgcagtgaaaagttaaatatccaagtacaccagccaatttcacacagtggaaccatgctgtcctcgggcacctgcacctcgcccaacagtcatcaactagatggaggctcctggctgcaaggaggatttgatgggaatgagtaaatgtgtcagcatagtccgtcccttctaatggaaaagcaacccaaagagcaaatcctattaatggctggatcagtatcatctacttgtcaaaaacattccatgaattatgagtcaaaattttatttatggtggcattacacacattaagagatgaggacttctgttagcataatttattagctggaaaagttgagaaggttctctggactcatttttataggtggaacctaagtgatctggataattgcccaccagcaaaattgctgggcatggtggacaaagaaaatgttccttctaatgattttttatgagctgagtagctattgttcccagctgagtgctcttttcctctttttattgttgctgagcaaaagaatttataaaaagctctttcttttgtattaaaaaccctgctcaattgaaatgcaagttcattaagtaatcttcatttctcttcctgccataataaccctttccctctctgttcgattcaacagtatctagcagcactgctccaaattttaagtctgaacagactatattacatagatgtagagaaatactcaatcttcagcattaagagggagcttaatttcacacgggtggaatatgatcactcaggctagatgttggccataaatttcaaattagtatctcaacttagcaggggggatcaacagtggcaaacttcaattatgacaggataaaaatcacatagagatattggttcaatatggacatctaaactataatgctaaaagccaataattagaataagttcattttaagaaaagcattaataatattagctaacgtttagtacctgtgccaaacattctacctatgttaccttgattttcatagccagcctaagaggtactattatgtatccccattttacaggttaagaaacaggctcagaggagtttaggatcttttccaagattacatagccagtaagtggtggcactaggaaccaaattcagactctgaatcgcatgctgtttatattatattgcactcattctaaatatgtgggaatcagaatgaaggggcttgtatgacttttggctcattttttgatgcatgtgacctgggattataaatgtgaaattaggtttacgaaaggatccagtgtcattgtgcatcatgggcaaggagtacctaatctctttaattcttccctggaagcttacgatgtccatccaagtgcacatagcaaaagttctgttgtaaagtttagcagagtgactttctttgactcagagtgatgacggaggaagctttgataagattttatctgaaatgttcatggacaagagctttcaaggagaacatccagagcaaggttctgaagacagctcatgaaggtgaagcagcagacctggcacaagaaatgaagagagagctcagtgtattaaagatgaaaacaagaaaaccgaatatattgaaaggagcagagaggcaatgaaaacaagacaactgaaatgaggtaacttgcagcaattgaaagggaatttcagtacttttatagaattcttaaaaattgtttcctgctgtttattttcaattttgaacagggttatttgtccatgccatactttttttgccaaattccaaaattgtgtatagttctatagttgtctggtggagtcaatggaactttagttaccagtctaagaatgtgtctttgagattgtccagttaattctctatttccagtagctgtaataaatggtgaaaaggtttctgactcctggagaaagtttctaactccttatgactaatattcataacagacttgtgagttccttgaacatggatacacctatatgcaagagtgtattccaaagctaactcagtgatctttccatttatctattcttggattagtggtgcctttgctctttccttctgtaaatgtgaatagttaagagttgactgcagaagtgtttacactttggcttccatgcctctggaatgtttgtgctttggtggtgagatgtgagactatatttgtatagtctgcatctctcaggctgccccagaatgttgtacagtgcagtgctgaagaaagcagcaggtacacacagaaatgcagcctttcctggttaaccctgcttggatctgagttacactttgtttcctgacttcttgggacttaggtaatcagtttgccttctactctatctcattttgtactcgcttacatactacattcttgtttgggctttcgtttcttcttgtaagcagagattttttaaaatccaatatgtgaaaatacggatgcactacaattaaataaataaaatgctgttgtgtttgttttgctttaaaattgtaaaggataaacaataagatagttttatctatgtggttttcccgatgcagttaaaataaaacctaatctgctaaaattgaa 412 TAZgctttccggcggttgcaccgggccggggtgccagcgcccgccttcccgtttcctcccgttccgcag(GenBank cgcgcccacggcctgtgaccccggcgaccgctccccagtgacgagagagcggggccgggcgcAccessiontgctccggcctgacctgcgaagggacctcggtccagtcccctgttgcgccgcgcccccgtccgtcc No.gtgcgcgggccagtcaggggccagtgtctcgagcggtcgaggtcgcagacctagaggcgccccNM_000116.5)acaggccggcccggggcgctgggagcgccggccgcgggccgggtggggatgcctctgcacgtgaagtggccgttccccgcggtgccgccgctcacctggaccctggccagcagcgtcgtcatgggcttggtgggcacctacagctgcttctggaccaagtacatgaaccacctgaccgtgcacaacagggaggtgctgtacgagctcatcgagaagcgaggcccggccacgcccctcatcaccgtgtccaatcaccagtcctgcatggacgaccctcatctctgggggatcctgaaactccgccacatctggaacctgaagttgatgcgttggacccctgcagctgcagacatctgcttcaccaaggagctacactcccacttcttcagcttgggcaagtgtgtgcctgtgtgccgaggagcagaatttttccaagcagagaatgaggggaaaggtgttctagacacaggcaggcacatgccaggtgctggaaaaagaagagagaaaggagatggcgtctaccagaaggggatggacttcattttggagaagctcaaccatggggactgggtgcatatcttcccagaagggaaagtgaacatgagttccgaattcctgcgtttcaagtggggaatcgggcgcctgattgctgagtgtcatctcaaccccatcatcctgcccctgtggcatgtcggaatgaatgacgtccttcctaacagtccgccctacttcccccgctttggacagaaaatcactgtgctgatcgggaagcccttcagtgccctgcctgtactcgagcggctccgggcggagaacaagtcggctgtggagatgcggaaagccctgacggacttcattcaagaggaattccagcatctgaagactcaggcagagcagctccacaaccacctccagcctgggagataggccttgcttgctgccttctggattcttggcccgcacagagctggggctgagggatggactgatgcttttagctcaaacgtggcttttagacagatttgttcatagaccctctcaagtgccctctccgagctggtaggcattccagctcctccgtgcttcctcagttacacaaaggacctcagctgcttctcccacttggccaagcagggaggaagaagcttaggcagggctctctttccttcttgccttcagatgttctctcccaggggctggcttcaggagggagcatagaaggcaggtgagcaaccagttggctaggggagcagggggcccaccagagctgtggagaggggaccctaagactcctcggcctggctcctacccaccgcccttgccgaaccaggagctgctcactacctcctcagggatggccgttggccacgtcttccttctgcctgagcttcccccccaccacaggccctttcctcaggcaaggtctggcctcaggtgggccgcaggcgggaaaagcagcccttggccagaagtcaagcccagccacgtggagcctagagtgagggcctgaggtctggctgcttgcccccatgctggcgccaacaacttctccatcctttctgcctctcaacatcacttgaatcctagggcctgggttttcatgtttttgaaacagaaccataaagcatatgtgttggcttgttgtaaaa 413ANGPTL3agaagaaaacagttccacgttgcttgaaattgaaaatcaagataaaaatgttcacaattaagctccttc(GenBanktttttattgttcctctagttatttcctccagaattgatcaagacaattcatcatttgattctctatctccagagAccessionccaaaatcaagatttgctatgttagacgatgtaaaaattttagccaatggcctccttcagttgggacatgNo.gtcttaaagactttgtccataagacgaagggccaaattaatgacatatttcaaaaactcaacatatttgaNM_014495.4)tcagtctttttatgatctatcgctgcaaaccagtgaaatcaaagaagaagaaaaggaactgagaagaactacatataaactacaagtcaaaaatgaagaggtaaagaatatgtcacttgaactcaactcaaaacttgaaagcctcctagaagaaaaaattctacttcaacaaaaagtgaaatatttagaagagcaactaactaacttaattcaaaatcaacctgaaactccagaacacccagaagtaacttcacttaaaacttttgtagaaaaacaagataatagcatcaaagaccttctccagaccgtggaagaccaatataaacaattaaaccaacagcatagtcaaataaaagaaatagaaaatcagctcagaaggactagtattcaagaacccacagaaatttctctatcttccaagccaagagcaccaagaactactccctttcttcagttgaatgaaataagaaatgtaaaacatgatggcattcctgctgaatgtaccaccatttataacagaggtgaacatacaagtggcatgtatgccatcagacccagcaactctcaagtttttcatgtctactgtgatgttatatcaggtagtccatggacattaattcaacatcgaatagatggatcacaaaacttcaatgaaacgtgggagaactacaaatatggttttgggaggcttgatggagaattttggttgggcctagagaagatatactccatagtgaagcaatctaattatgttttacgaattgagttggaagactggaaagacaacaaacattatattgaatattctttttacttgggaaatcacgaaaccaactatacgctacatctagttgcgattactggcaatgtccccaatgcaatcccggaaaacaaagatttggtgttttctacttgggatcacaaagcaaaaggacacttcaactgtccagagggttattcaggaggctggtggtggcatgatgagtgtggagaaaacaacctaaatggtaaatataacaaaccaagagcaaaatctaagccagagaggagaagaggattatcttggaagtctcaaaatggaaggttatactctataaaatcaaccaaaatgttgatccatccaacagattcagaaagctttgaatgaactgaggcaaatttaaaaggcaataatttaaacattaacctcattccaagttaatgtggtctaataatctggtattaaatccttaagagaaagcttgagaaatagattttttttatcttaaagtcactgtctatttaagattaaacatacaatcacataaccttaaagaataccgtttacatttctcaatcaaaattcttataatactatttgttttaaattttgtgatgtgggaatcaattttagatggtcacaatctagattataatcaataggtgaacttattaaataacttttctaaataaaaaatttagagacttttattttaaaaggcatcatatgagctaatatcacaactttcccagtttaaaaaactagtactcttgttaaaactctaaacttgactaaatacagaggactggtaattgtacagttcttaaatgttgtagtattaatttcaaaactaaaaatcgtcagcacagagtatgtgtaaaaatctgtaatacaaatttttaaactgatgcttcattttgctacaaaataatttggagtaaatgtttgatatgatttatttatgaaacctaatgaagcagaattaaatactgtattaaaataagttcgctgtctttaaacaaatggagatgactactaagtcacattgactttaacatgaggtatcactataccttatttgttaaaatatatactgtatacattttatatattttaacacttaatactatgaaaacaaataattgtaaaggaatcttgtcagattacagtaagaatgaacatatttgtggcatcgagttaaagtttatatttcccctaaatatgctgtgattctaatacattcgtgtaggttttcaagtagaaataaacctcgtaacaagttactgaacgtttaaacagcctgacaagcatgtatatatgtttaaaattcaataaacaaagacccagtccctaaattatagaaatttaaattattcttgcatgtttatcgacatcacaacagatccctaaatccctaaatccctaaagattagatacaaattttttaccacagtatcacttgtcagaatttatttttaaatatgattttttaaaactgccagtaagaaattttaaattaaacccatttgttaaaggatatagtgcccaagttatatggtgacctacctttgtcaatacttagcattatgtatttcaaattatccaatatacatgtcatatatatttttatatgtcacatatataaaagatatgtatgatctatgtgaatcctaagtaaatattttgttccagaaaagtacaaaataataaaggtaaaaataatctataattttcaggaccacagactaagctgtcgaaattaacgctgatttttttagggccagaataccaaaatggctcctctcttcccccaaaattggacaatttcaaatgcaaaataattcattatttaatatatgagttgcttcctctatttggtttcc 414 DGAT2tgccccgttgtgaggtgataaagtgttgcgctccgggacgccagcgccgcggctgccgcctctgct(GenBankggggtctaggctgtttctctcgcgccaccactggccgccggccgcagctccaggtgtcctagccgcAccessionccagcctcgacgccgtcccgggacccctgtgctctgcgcgaagccctggccccgggggccggg No.gcatgggccaggggcgcggggtgaagcggcttcccgcggggccgtgactgggcgggcttcagcNM_001253891.1)catgaagaccctcatagccgcctactccggggtcctgcgcggcgagcgtcaggccgaggctgaccggagccagcgctctcacggaggacctgcgctgtcgcgcgaggggtctgggagatggggagtggcctgcagtgccatcctcatgtacatattctgcactgattgctggctcatcgctgtgctctacttcacttggctggtgtttgactggaacacacccaagaaaggtggcaggaggtcacagtgggtccgaaactgggctgtgtggcgctactttcgagactactttcccatccagctggtgaagacacacaacctgctgaccaccaggaactatatctttggataccacccccatggtatcatgggcctgggtgccttctgcaacttcagcacagaggccacagaagtgagcaagaagttcccaggcatacggccttacctggctacactggcaggcaacttccgaatgcctgtgttgagggagtacctgatgtctggaggtatctgccctgtcagccgggacaccatagactatttgctttcaaagaatgggagtggcaatgctatcatcatcgtggtcgggggtgcggctgagtctctgagctccatgcctggcaagaatgcagtcaccctgcggaaccgcaagggctttgtgaaactggccctgcgtcatggagctgacctggttcccatctactcctttggagagaatgaagtgtacaagcaggtgatcttcgaggagggctcctggggccgatgggtccagaagaagttccagaaatacattggtttcgccccatgcatcttccatggtcgaggcctcttctcctccgacacctgggggctggtgccctactccaagcccatcaccactgttgtgggagagcccatcaccatccccaagctggagcacccaacccagcaagacatcgacctgtaccacaccatgtacatggaggccctggtgaagctcttcgacaagcacaagaccaagttcggcctcccggagactgaggtcctggaggtgaactgagccagccttcggggccaattccctggaggaaccagctgcaaatcacttttttgctctgtaaatttggaagtgtcatgggtgtctgtgggttatttaaaagaaattataacaattttgctaaaccattacaatgttaggtcttttttaagaaggaaaaagtcagtatttcaagttctttcacttccagcttgccctgttctaggtggtggctaaatctgggcctaatctgggtggctcagctaacctctcttcttcccttcctgaagtgacaaaggaaactcagtcttcttggggaagaaggattgccattagtgacttggaccagttagatgattcactttttgcccctagggatgagaggcgaaagccacttctcatacaagcccctttattgccactaccccacgctcgtctagtcctgaaactgcaggaccagtttctctgccaaggggaggagttggagagcacagttgccccgttgtgtgagggcagtagtaggcatctggaatgctccagtttgatctcccttctgccacccctacctcacccctagtcactcatatcggagcctggactggcctccaggatgaggatgggggtggcaatgacaccctgcaggggaaaggactgccccccatgcaccattgcagggaggatgccgccaccatgagctaggtggagtaactggtttttcttgggtggctgatgacatggatgcagcacagactcagccttggcctggagcacatgcttactggtggcctcagtttaccttccccagatcctagattctggatgtgaggaagagatccctcttcagaaggggcctggccttctgagcagcagattagttccaaagcaggtggcccccgaacccaagcctcacttttctgtgccttcctgagggggttgggccggggaggaaacccaaccctctcctgtgtgttctgttatctcttgatgagatcattgcaccatgtcagacttttgtatatgccttgaaaataaatgaaagtgagaatcctctaaaaaaaaaaaa 596 HBVctccaccactttccaccaaactcttcaagatcccagagtcagggccctgtactttcctgctggtggctcGenbankaagttccggaacagtaaaccctgctccgactactgcctctcccatatcgtcaatcttctcgaggactgAccessiongggaccctgtaccgaatatggagagcaccacatcaggattcctaggacccctgctcgtgttacagg No.cggggtttttcttgttgacaagaatcctcacaataccacagagtctagactcgtggtggacttctctcaKC315400.1attttctagggggagcacccacgtgtcctggccaaaatttgcagtccccaacctccaatcactcaccaacctcttgtcctccaatttgtcctggttatcgctggatgtgtctgcggcgttttatcatcttcctcttcatcctgctgctatgcctcatcttcttgttggttcttctggactaccaaggtatgttgcccgtttgtcctctacttccaggaacatcaactaccagcaccggaccatgcaaaacctgcacaactactgctcaagggacctctatgtttccctcatgttgctgtacaaaacctacggacggaaactgcacctgtattcccatcccatcatcttgggctttcgcaaaatacctatgggagtgggcctcagtccgtttctcttggctcagtttactagtgccatttgttcagtggttcgtagggattcccccactgtctggctttcagttatatggatgatgtggttttgggggccaagtctgtacaacatcttgagtccctttataccgctgttaccaattttcttttatctttgggtatacatttaaaccctcacaaaacaaaaagatggggatattcccttaacttcatgggatatgtaattgggagttggggcactttgcctcaggaacatattgtacaaaaaatcaagcaatgttttaggaaacttcctgtaaacaggcctattgattggaaagtatgtcaacraattgtgggtcttttggggtttgccgcccctttcacgcaatgtggatatcctgctttaatgcctttatatgcatgtatacaagctaagcaggcttttactttctcgccaacttacaaggcctttctgtgtaaacaatatctgaacctttaccccgttgctcggcaacggtcaggtctttgccaagtgtttgctgacgcaacccccactggttggggcttggccataggccatcagcgcatgcgtggaacctttgtggctcctctgccgatccatactgcggaactcctagcagcttgttttgctcgcagccggtctggagcaaaacttatcggcaccgacaactctgttgtcctctctcggaaatacacctcctttccatggctgctaggatgtgctgccaactggatcctgcgcgggacgtcctttgtctacgtcccgtcggcgctgaatcccgcggacgacccatctcggggccgtttgggactctaccgtccccttctgcgtctgccgttccgcccgaccacggggcgcacctctctttacgcggtctccccgtctgtgccttctcatctgccggaccgtgtgcacttcgcttcacctctgcacgtcgcatggagaccaccgtgaacgcccacgggaacctgcccaaggtcttgcataagaggactcttggactttcagcaatgtcaacgaccgaccttgaggcatacttcaaagactgtgtgtttactgagtgggaggagttgggggaggaggttaggttaaaggtctttgtactaggaggctgtaggcataaattggtgtgttcaccagcaccatgcaactttttcacctctgcctaatcatctcatgttcatgtcctactgttcaagcctccaagctgtgccttgggtggctttggggcatggacattgacccgtataaagaatttggagcttctgtggagttactctattttttgccttctgacttctttccttctattcgagatctcctcgacaccgcctctgctctgtatcgggaggccttagagtctccggaacattgttcacctcaccatacggcactcaggcaagcaattctgtgttggggtgagttaatgaatctagccacctgggtgggaagtaatttggaagatccagcatccagggaattagtagtcagctatgtcaacgttaatatgggcctaaaaatcagacaactattgtggtttcacatttcctgtcttacttttgggagagaaactgttcttgaatatttggtgtcttttggagtgtggattcgcactcctcctgcatatagaccacaaaatgcccctatcttatcaacacttccggaaactactgttgttagacgaagaggcaggtcccctagaagaagaactccctcgcctcgcagacgaaggtctcaatcgccgcgtcgcagaagatctcaatctcgggaatctcaatgttagtattccttggacacataaggtgggaaactttacggggctttattcttctacggtaccttgctttaatcctaaatggcaaactccttcttttcctgacattcatttgcaggaggacattgttgatagatgtaagcaatttgtggggccccttacagtaaatgaaaacaggagacttaaattaattatgcctgctaggttttatcccaatgttactaaatatttgcccttagataaagggatcaaaccgtattatccagagtatgtagttaatcattacttccagacgcgacattatttacacactctttggaaggcggggatcttatataaaagagagtccacacgtagcgcctcattttgcgggtcaccatattcttgggaacaagatctacagcatgggaggttggtcttccaaacctcgaaaaggcatggggacaaatctttctgtccccaatcccctgggattcttccccgatcatcagttggaccctgcattcaaagccaactcagaaaatccagattgggacctcaacccacacaaggacaactggccggacgccaacaaggtgggagtgggagcattcgggccagggttcacccctcctcatgggggactgttggggtggagccctcaggctcagggcatattcacaacagtgccagcagctcctcctcctgcctccaccaatcggcagtcaggaaggcagcctactcccttctctccacctctaagagacactcatcctcaggccatgcagtggaa 534 ASO 1GalNAc4-ps-GalNAc4-ps-GalNAc4-po-mA-po-lnGpslnApslnTpslnApslnApsApsAps(5OH)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m)C 535 ASO 2 mA-po-lnGpslnApslnTpslnApslnApsApsAps(5OH)CpsGps(5m)Cps(5m)CpsGps(5m)CpslnApslnGpslnApscp(5m)C ⁺ln = Locked nucleic acid (LNA) =

lnA = Locked nucleic acid (LNA) A; ln(5m)C = ln(5m)C = Locked nucleicacid (LNA)-5 methyl C; lnG = Locked nucleic acid (LNA) G; lnT = Lockednucleic acid (LNA) T; (5m)C = 5 methyl C; cp = scp = cyclopropyl; cpC =scpC = cyclopropyl C; scp(5m)C = cyclopropyl-5 methyl C; (5OH)C =

po = phosphodiester linkage; ps = phosphorothioate linkage

TABLE 6 siNA Activity HepG2.2.15 HepG2.2.15 Sense Strand AntisenseStrand in vitro in vitro ds-siNA ID SEQ ID NO. SEQ ID NO. EC50* CC50(nM) ds-siNA-001 307 363 A >40 ds-siNA-002 308 364 A >40 ds-siNA-003 309365 B >40 ds-siNA-004 310 366 B >40 ds-siNA-005 311 367 B >40ds-siNA-006 312 368 C >40 ds-siNA-007 313 369 A >40 ds-siNA-008 314 370A >40 ds-siNA-009 315 371 B >40 ds-siNA-010 316 372 A >40 ds-siNA-011317 373 B >40 ds-siNA-012 318 374 A >40 ds-siNA-013 319 375 A >40ds-siNA-014 320 376 B >40 ds-siNA-015 321 377 A >40 ds-siNA-016 322 377C >40 ds-siNA-017 323 377 A >40 ds-siNA-018 324 378 A >40 ds-siNA-019325 378 A >40 ds-siNA-020 326 379 A >40 ds-siNA-021 327 379 B >40ds-siNA-022 328 380 A >40 ds-siNA-023 329 380 B >40 ds-siNA-024 330 381A >40 ds-siNA-025 331 382 A >40 ds-siNA-026 332 383 C >40 ds-siNA-027333 384 A >40 ds-siNA-028 334 385 B >40 ds-siNA-029 335 386 A >40ds-siNA-030 336 387 C >40 ds-siNA-031 337 388 A >40 ds-siNA-032 338 388C >40 ds-siNA-033 339 389 B >40 ds-siNA-034 340 389 C >40 ds-siNA-035341 390 A >40 ds-siNA-036 342 391 A >40 ds-siNA-037 343 392 B >40ds-siNA-038 344 393 A >40 ds-siNA-039 345 394 A >40 ds-siNA-040 346 395A >40 ds-siNA-041 347 396 C >40 ds-siNA-042 348 397 A >40 ds-siNA-043349 398 B >40 ds-siNA-044 350 399 A >40 ds-siNA-045 351 400 A >40ds-siNA-046 352 401 A >40 ds-siNA-047 353 402 A >40 ds-siNA-048 354 403A >40 ds-siNA-049 355 404 B >40 ds-siNA-050 356 405 A >40 ds-siNA-051357 406 A >40 ds-siNA-052 358 406 A >40 ds-siNA-053 359 407 A >40ds-siNA-054 360 407 A >40 ds-siNA-055 361 408 A >40 ds-siNA-056 362 409A >40 ds-siNA-0164 423 482 *A = EC50 < 0.5 nM; B = 0.5 nM < EC50 < 1; C= EC50 > 1 nm

TABLE 10 siNA Activity Sense Antisense Strand Strand Max HBsAg SEQ 3′Ligand SEQ HepG2.2.15 HepG2.2.15 Knock Down ds-siNA ID ID NO Monomer⁺ IDNO EC50* CC50 (nM) (Log₁₀)** ds-siNA-057 415 p-(PS)2-GalNac4 445 ND ND Xds-siNA-058 415 p-(PS)2-GalNac4 446 ND ND X ds-siNA-059 415p-(PS)2-GalNac4 447 ND ND Y ds-siNA-060 416 p-(PS)2-GalNac4 448 ND ND Yds-siNA-061 416 p-(PS)2-GalNac4 449 ND ND Y ds-siNA-062 416p-(PS)2-GalNac4 450 ND ND Y ds-siNA-063 416 p-(PS)2-GalNac4 451 ND ND Xds-siNA-064 417 5′-GalNAc4- 452 ND ND Y (PS)2-p-TEG-p ds-siNA-065 4175′-GalNAc4- 452 ND ND Y (PS)2-p-HEG-p ds-siNA-066 417 5′-GalNAc4- 452 NDND Y (PS)2-p-(HEG-p)2 ds-siNA-067 417 5′-GalNAc4- 452 ND ND Z(PS)2-p-(HEG-p)2 ds-siNA-068 418 p-(PS)2-GalNac4 453 ND ND Y ds-siNA-069418 p-(PS)2-GalNac4 454 ND ND Y ds-siNA-070 419 p-(PS)2-GalNac4 455 NDND Y ds-siNA-071 419 p-(PS)2-GalNac4 456 ND ND Y ds-siNA-072 420p-(PS)2-GalNac4 457 ND ND X ds-siNA-073 421 p-(PS)2-GalNac4 458 ND ND Xds-siNA-074 422 p-(PS)2-GalNac4 459 ND ND Y ds-siNA-075 423p-(PS)2-GalNac4 460 ND ND Y ds-siNA-076 423 p-(PS)2-GalNac4 461 ND ND Yds-siNA-077 423 5′-GalNAc4- 458 ND ND Y (PS)2-p-TEG-p ds-siNA-078 4235′-GalNAc4- 458 ND ND X (PS)2-p-HEG-p ds-siNA-079 423 5′-GalNAc4- 458 NDND Y (PS)2-p-(HEG-p)2 ds-siNA-080 423 p-(PS)2-GalNac4 462 ND ND Xds-siNA-081 423 p-(PS)2-GalNac4 463 ND ND X ds-siNA-082 423p-(PS)2-GalNac4 447 ND ND X ds-siNA-083 423 5′-GalNAc4- 458 ND ND Z(PS)2-p-(HEG-p)2 ds-siNA-084 423 p-(PS)2-GalNac4 457 ND ND X ds-siNA-085423 p-(PS)2-GalNac4 464 ND ND X ds-siNA-086 423 p-(PS)2-GalNac4 465 NDND X ds-siNA-087 423 p-(PS)2-GalNac4 466 ND ND Y ds-siNA-088 423p-(PS)2-GalNac4 467 ND ND Z ds-siNA-089 423 p-(PS)2-GalNac4 468 ND ND Zds-siNA-090 423 p-(PS)2-GalNac4 469 B >1000 X ds-siNA-091 423p-(PS)2-GalNac4 470 C >1000 ND ds-siNA-092 423 p-(PS)2-GalNac4 471B >1000 ND ds-siNA-093 423 p-(PS)2-GalNac4 472 B >1000 ND ds-siNA-094423 p-(PS)2-GalNac4 473 B >1000 ND ds-siNA-095 423 p-(PS)2-GalNac4 474C >1000 ND ds-siNA-096 423 p-(PS)2-GalNac4 475 B >1000 ND ds-siNA-097423 p-(PS)2-GalNac4 476 B >1000 ND ds-siNA-098 423 p-(PS)2-GalNac4 477A >1000 ND ds-siNA-099 423 p-(PS)2-GalNac4 478 B >1000 ND ds-siNA-0100423 p-(PS)2-GalNac4 479 B >1000 ND ds-siNA-0101 423 p-(PS)2-GalNac4 480B >1000 ND ds-siNA-0102 423 p-(PS)2-GalNac4 481 A >1000 ND ds-siNA-0103423 p-(PS)2-GalNac4 482 ND ND ND ds-siNA-0104 423 p-(PS)2-GalNac4 483 NDND ND ds-siNA-0105 423 p-(PS)2-GalNac4 458 ND ND Z ds-siNA-0106 423p-(PS)2-GalNac4 458 ND ND Y ds-siNA-0107 424 p-(PS)2-GalNac4 457 ND ND Xds-siNA-0108 424 p-(PS)2-GalNac4 484 ND ND X ds-siNA-0109 424p-(PS)2-GalNac4 485 ND ND X ds-siNA-0110 425 p-(PS)2-GalNac4 486 ND NDND ds-siNA-0111 425 p-(PS)2-GalNac4 487 ND ND ND ds-siNA-0112 425p-(PS)2-GalNac4 488 ND ND ND ds-siNA-0113 426 p-(PS)2-GalNac4 489 ND NDND ds-siNA-0114 427 p-(PS)2-GalNac4 490 ND ND X ds-siNA-0115 428p-(PS)2-GalNac4 491 ND ND Y ds-siNA-0116 429 p-(PS)2-GalNac4 492 ND NDND ds-siNA-0117 429 p-(PS)2-GalNac4 493 ND ND ND ds-siNA-0118 429p-(PS)2-GalNac4 494 ND ND ND ds-siNA-0119 430 p-(PS)2-GalNac4 495 ND NDX ds-siNA-0120 430 p-(PS)2-GalNac4 496 ND ND ND ds-siNA-0121 431p-(PS)2-GalNac4 497 ND ND Y ds-siNA-0122 432 p-(PS)2-GalNac4 498 ND NDND ds-siNA-0123 432 p-(PS)2-GalNac4 500 ND ND ND ds-siNA-0124 433p-(PS)2-GalNac4 501 ND ND ND ds-siNA-0125 434 p-(PS)2-GalNac4 502 ND NDY ds-siNA-0126 435 p-(PS)2-GalNac4 502 ND ND Y ds-siNA-0127 435p-(PS)2-GalNac4 503 ND ND X ds-siNA-0128 435 p-(PS)2-GalNac4 501 ND ND Xds-siNA-0129 435 p-(PS)2-GalNac4 504 ND ND Y ds-siNA-0130 435p-(PS)2-GalNac4 505 ND ND Z ds-siNA-0131 435 p-(PS)2-GalNac4 506 ND ND Yds-siNA-0132 435 p-(PS)2-GalNac4 507 ND ND Z ds-siNA-0133 435p-(PS)2-GalNac4 508 ND ND Z ds-siNA-0134 435 p-(PS)2-GalNac4 509 ND ND Zds-siNA-0135 435 p-(PS)2-GalNac4 510 ND ND Y ds-siNA-0136 435p-(PS)2-GalNac4 511 B >1000 ND ds-siNA-0137 435 p-(PS)2-GalNac4 512B >1000 ND ds-siNA-0138 435 p-(PS)2-GalNac4 513 A >1000 ND ds-siNA-0139435 p-(PS)2-GalNac4 514 B >1000 ND ds-siNA-0140 435 p-(PS)2-GalNac4 515C >1000 ND ds-siNA-0141 435 p-(PS)2-GalNac4 516 A >1000 ND ds-siNA-0142435 p-(PS)2-GalNac4 517 C >1000 ND ds-siNA-0143 435 p-(PS)2-GalNac4 518C >1000 ND ds-siNA-0144 435 p-(PS)2-GalNac4 519 ND ND ND ds-siNA-0145436 p-(PS)2-GalNac4 520 ND ND ND ds-siNA-0146 437 p-(PS)2-GalNac4 502 NDND Y ds-siNA-0147 438 p-(PS)2-GalNac4 501 ND ND X ds-siNA-0148 438p-(PS)2-GalNac4 521 ND ND X ds-siNA-0149 438 p-(PS)2-GalNac4 522 ND ND Xds-siNA-0150 439 p-(PS)2-GalNac4 523 ND ND X ds-siNA-0151 440p-(PS)2-GalNac4 524 ND ND Y ds-siNA-0152 441 p-(PS)2-GalNac4 525 ND ND Yds-siNA-0153 441 p-(PS)2-GalNac4 526 ND ND X ds-siNA-0154 441p-(PS)2-GalNac4 527 ND ND ND ds-siNA-0155 442 p-(PS)2-GalNac4 525 ND NDX ds-siNA-0156 442 p-(PS)2-GalNac4 528 ND ND Y ds-siNA-0157 442p-(PS)2-GalNac4 529 ND ND ND ds-siNA-0158 443 p-(PS)2-GalNac4 458 ND NDY ds-siNA-0159 444 p-(PS)2-GalNac4 502 ND ND Y ds-siNA-0160 423p-(PS)2-GalNac4 458 ND ND ND ds-siNA-0161 533 p-(PS)2-GalNac4 489 ND NDND ds-siNA-0162 534 p-(PS)2-GalNac4 491 ND ND ND ds-siNA-0163 432p-(PS)2-GalNac4 496 ND ND ND ds-siNA-0165 435 p-(PS)2-GalNac4 502 ND NDND ds-siNA-0166 442 p-(PS)2-GalNac4 530 ND ND ND ds-siNA-0167 427p-(PS)2-GalNAc4 491 ND ND ND ds-siNA-0168 439 p-(PS)2-GalNac4 531 ND NDND ds-siNA-0169 423 p-(PS)2-GalNac4 532 ND ND ND ds-siNA-0170 441p-(PS)2-GalNAc4 530 ND ND ND ds-siNA-0171 442 p-(PS)2-GalNac4 533 ND NDND ds-siNA-0172 424 p-(PS)2-GalNac4 536 A   >1 ND ds-siNA-0173 438 None537 ds-siNA-0174 438 None 538 ds-siNA-0175 438 None 501 ds-siNA-0176 438p-(PS)2-GalNAc4 537 ds-siNA-0177 438 p-(PS)2-GalNAc4 538 ds-siNA-0178438 p-(PS)2-GalNAc4 539 ⁺Ligand monomers are attached to the 3′ end ofthe sense strand, unless the ligand monomer is annotated with 5′, inwhich the ligand monomer is attached to the 5′ end of the sense strand.Linkers are represented as p-(PS)2, (PS)2-p-TEG-p, (PS)2-p-HEG-p, or(PS)2-p-(HEG-p)2. *For EC50, A = EC50 ≤ 5 nM; B = 5 nM < EC50 < 10; C =EC50 ≥ 10. **For Max HBsAg knock down, X ≥1 log₁₀ reduction in HBsAg, Yis 0.5-1 log₁₀ reduction in HBsAg, and Z is <0.5 log₁₀ reduction inHBsAg.

What is claimed is:
 1. A method of treating hepatitis B virus (HBV)comprising administering to a subject with HBV a double stranded shortinterfering nucleic acid (siNA) comprising: (a) a sense strandcomprising 19-21 nucleotides in a nucleic acid sequence that is at least80% identical to SEQ ID NO: 40, wherein 15 or more of the nucleotidesare modified nucleotides independently selected from a 2′-O-methylnucleotide and a 2′-fluoro nucleotide, and wherein at least 11 of themodified nucleotides are a 2′-O-methyl nucleotide and at least 4 of themodified nucleotides are a 2′-fluoro nucleotide; and (b) an antisensestrand comprising 19-21 nucleotides in a nucleic acid sequence that isat least 80% complementary to SEQ ID NO: 40, wherein 15 or more of thenucleotides are modified nucleotides independently selected from a2′-O-methyl nucleotide and a 2′-fluoro nucleotide, and wherein at least11 of the modified nucleotides are a 2′-O-methyl nucleotide and 4 to 6of the modified nucleotides are a 2′-fluoro nucleotide.
 2. The method ofclaim 1, wherein: (a) the nucleotide(s) at position 3, 5, 7, 8, 9, 10,11, 12, 14, 17, or 19 from the 5′ end of the sense strand is a 2′-fluoronucleotide; and (b) the nucleotide(s) at position 2, 5, 6, 8, 10, 14,16, 17, or 18 from the 5′ end of the antisense strand is a 2′-fluoronucleotide.
 3. The method of claim 1, wherein the sense strand comprisesSEQ ID NO: 438 or SEQ ID NO: 435, and the antisense strand comprises anyone of SEQ ID NOs: 501-519, SEQ ID NO: 537, SEQ ID NO: 538, or SEQ IDNO:
 539. 4. The method of claim 1, further comprising aN-acetylgalactosamine (GalNAc) attached to the 3′ of the sense strand.5. A method of treating hepatitis B virus (HBV) comprising administeringto a subject with HBV a double stranded short interfering nucleic acid(siNA) comprising: (a) a sense strand comprising 19 nucleotides, wherein2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of thesense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4,6, and 10-19 from the 5′ end of the sense strand; and (b) an antisensestrand comprising 21 nucleotides, wherein 2′-fluoro nucleotides are atpositions 2, 6, 14, and 16 from the 5′ end of the antisense strand, andwherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and17-21 from the 5′ end of the antisense strand.
 6. The method of claim 5further comprising (i) the nucleotides at positions 1 and 2 andpositions 2 and 3 from the 5′ end of the sense strand are connected byphosphorothioate internucleoside linkages; and (ii) the nucleotides atpositions 1 and 2; positions 2 and 3; positions 19 and 20; and positions20 and 21 from the 5′ end of the antisense strand are connected byphosphorothioate internucleoside linkages.
 7. The method of claim 5further comprising a galactosamine attached to the sense strand.
 8. Themethod of claim 7, wherein the conjugated moiety isN-acetylgalactosamine (GalNAc).
 9. The method of claim 5, wherein thesense strand comprises SEQ ID NO: 438, and the antisense strandcomprises any one of SEQ ID NO: 501, SEQ ID NO: 537, SEQ ID NO: 538, orSEQ ID NO:
 539. 10. The method of claim 9, wherein the senses strandcomprises SEQ ID NO: 438 and the antisense comprises SEQ ID NO:
 501. 11.The method of claim 10, wherein the conjugated moiety isN-acetylgalactosamine (GalNAc).
 12. The method of claim 11, wherein theGalNAc comprises a structure of Formula (VII):

wherein n is 1, and R is OH.
 13. The method of claim 5, wherein thesense strand is at least 80% identical to SEQ ID NO: 40 and theantisense strand is at least 80% complementary to SEQ ID NO:
 40. 14. Themethod of claim 5, wherein the sense strand, the antisense strand, orboth comprises at least one overhang consisting of 1 or 2 nucleotides.15. A method of treating hepatitis B virus (HBV) comprisingadministering to a subject with HBV a double stranded short interferingnucleic acid (siNA) comprising: (a) a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an anti sense strand of5′-mApsfUJpsmUmGmAfGmAmGmAmAmGmUmCfCmAfCmCmAmCpsmG psmA-3′ (SEQ ID NO:501); (b) a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an anti sense strand of 5′-mApsfUpsmUmGmAfGmAfGfAmAmGmUmCfCmAfCmCmAmCpsmGps mA-3′ (SEQ ID NO: 521); (c) a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GaaNAc-3′ (SEQ IDNO: 438) and an antitsense strand of5′-mApsfUpsmUmGfAmGmAfGmAmAmGmUmCfCmAmCfCmAmCpsmGp smA-3′ (SEQ ID NO:522); (d) a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsf4PpsmUmGmAfGmAmGmAmAmGmUmCfCmAfCmCmAmCpsmG psmA-3′ (SEQ ID NO:537); (e) a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCf2PmAfCmCmAmCpsmG psmA-3′ (SEQ ID NO:538); or (f) a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsfJpsmUmGmAfGmAmGmAmAmGmUmCfCmAfXmCmAmCpsmG psmA-3′ (SEQ ID NO:539), wherein fX is

wherein GalNAc comprises a structure of Formula (VII):

wherein n is 1, and R is OH.
 16. The method of claim 15, comprising asense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCfCmAfCmCmAmCpsmGpsmA-3′ (SEQ ID NO:501).
 17. The method of claim 15, comprising a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsfUpsmUmGmAfGmAfGfAmAmGmUmCfCmAfCmCmAmCpsmGpsmA-3′ (SEQ ID NO:521).
 18. The method of claim 15, comprising a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsfUpsmUmGfAmGmAfGmAmAmGmUmCfCmAmCfCmAmCpsmGpsmA-3′ (SEQ ID NO:522).
 19. The method of claim 15, comprising a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsf4PpsmUmGmAfGmAmGmAmAmGmUmCfCmAfCmCmAmCpsmGpsmA-3′ (SEQ ID NO:537).
 20. The method of claim 15, comprising a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCf2PmAfCmCmAmCpsmGpsmA-3′ (SEQ ID NO:538).
 21. The method of claim 15, comprising a sense strand of5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ IDNO: 438) and an antisense strand of5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCfCmAfXmCmAmCpsmGpsmA-3′ (SEQ ID NO:539), wherein fX is


22. A method of treating hepatitis B virus (HBV) comprisingadministering to a subject with HBV a short interfering nucleic acid(siNA) molecule comprising: (a) a sense strand comprising a nucleic acidsequence consisting of SEQ ID NO: 438 or SEQ ID NO: 435, and (b) anantisense strand comprising a nucleic acid sequence consisting of anyone of SEQ ID NO: 501, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 537,SEQ ID NO: 538, and SEQ ID NO:
 539. 23. The method of claim 22 furthercomprising a N-acetylgalactosamine (GalNAc) attached to the 3′ of thesense strand.
 24. The method of claim 22, wherein the GalNAc comprises astructure of Formula (VII):

wherein n is 1, and R is OH.
 25. The method of claim 22, wherein thesenses strand comprises a nucleic acid sequence consisting of SEQ ID NO:438 and the antisense comprises a nucleic acid sequence consisting ofSEQ ID NO:
 501. 26. The method of claim 25 further comprising aN-acetylgalactosamine (GalNAc) attached to the 3′ of the sense strand.27. The method of claim 26, wherein the GalNAc comprises a structure ofFormula (VII):

wherein n is 1, and R is OH.
 28. The method of claim 25, wherein: (a)the nucleotide(s) at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, or 19from the 5′ end of the sense strand is a 2′-fluoro nucleotide; (b) thenucleotide(s) at position 2, 5, 6, 8, 10, 14, 16, 17, or 18 from the 5′end of the antisense strand is a 2′-fluoro nucleotide; (c) thenucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ endof the sense strand are connected by phosphorothioate internucleosidelinkages; and (d) the nucleotides at positions 1 and 2; positions 2 and3; positions 19 and 20; and positions 20 and 21 from the 5′ end of theantisense strand are connected by phosphorothioate internucleosidelinkages.
 29. The method of claim 28 further comprising aN-acetylgalactosamine (GalNAc) attached to the 3′ of the sense strand.30. The method of claim 29, wherein the GalNAc comprises a structure ofFormula (VII):

wherein n is 1, and R is OH.